Latest revision as of 03:08, 22 October 2019

Notebook

Lab Notebook

Milestones

Lab Notebook

Here you can find short summaries of all the work we did in the lab. You can click on the boxes for more information. More information about our achievements outside the lab is shown on the Milestones page.

June

Monday June 17, 2019 - Stocks

Tuesday June 18, 2019 - Transformation

Wednesday June 19, 2019 - Small culture

Thursday June 20, 2019 - Miniprep & protein expression

Friday June 21, 2019 - Digestion & protein pellets

Monday June 24, 2019 - Ligation & column preparation

Tuesday June 25, 2019 - Purification & small culture

Wednesday June 26, 2019 - Miniprep & SDS-PAGE

Thursday June 27, 2019 - SDS-PAGE, sequencing & new LB stock

Friday June 28, 2019 - 2^nd Transformation Sbit, IPTG stock & New plan expression

Since the first round of expression and purification of the dCas9-NL_Sbit protein was unfortunately unsuccessful, we adapted the expression and purification protocol to now test 4 different sets of expression conditions and 2 different lysis methods. To start the second trial round of protein expression of our sensor we again transformed our plasmid into E. coli BL21 (DE3).

For transformation, a 4x dilution of the miniprepped plasmid DNA was prepared, taking 1 μl from plasmid tube 1 and adding together with 3 μl in another tube. Final plasmid concentration ~50 ng/μl. 1 μl of this was added to the bacteria, the remainder was placed in the freezer. The bacteria were heat-shocked for 40 s. 100 μl of the final LB/bacteria solution was spread out over an agar plate and left to incubate at room temperature over the weekend.

Also, new IPTG stock solution was prepared. We added 6.7 ml MQ water to 1.6 g IPTG, totalling 8.0 ml solution (in hind sight the total solution should have been 6.7 ml to obtain 1M conc.), resulting in a 0.84M stock. Hence, for 1mM final concentration, add 1.2 ml stock IPTG to 1L medium. For 200nM final concentration, add 0.25 ml stock IPTG.

New plan Expression

From the SDS-PAGE results we concluded that no dCas9-Sbit was formed. Therefore a new plan was made to test different combinations of expression conditions based on information we gathered. The plan is to test four different condition combinations.

The following conditions will be the same:

Host: BL21(DE3)
Medium: 750 mL LB
OD600: 0.5
Half of the culture will be lysed with ultrasound, the other half with Homogenizer

July

Monday July 1, 2019 - Small culture

Tuesday July 2, 2019 - Small culture

Wednesday July 3, 2019 - Expression

Split-NL

As the initial attempt of expressing the dCas9-NL-Sbit fusion protein failed, we now try 2 sets of different expression conditions in parallel (we narrowed down to only 2 condition sets, in contrast to the 4 we planned to do initially to save work and time, while still using the most interesting conditions). Namely:

	[IPTG]	Temperature (°C)	Color label
1.	0.2 mM	16	Green
2.	1 mM	16	Blue

Both cultures were at 750 mL scale, with 0.75 mL 1000x IPTG added. The cultures were grown at 37 °C, 160 rpm until reaching OD₆₀₀ ~0.5-0.6, upon which IPTG was added according to the scheme below.

	Color label	[IPTG]	Add this volume of 0.84M IPTG stock solution to 750 ml medium
1.	Green	0.2 mM	0.18 ml
2.	Blue	1 mM	0.90 ml

The cultures were the incubated overnight at 16°C, 160 rpm.

Thursday July 4, 2019 - Centrifugation

Friday July 5, 2019 - Sonication lysis & protein purification

Monday July 8, 2019 - SDS-PAGE

Tuesday July 9, 2019 - SDS-PAGE

Wednesday July 10, 2019 - Denaturing His-tag purification

Thursday July 11, 2019 - Denaturation

Friday July 12, 2019 - Design

Monday July 15, 2019 - General

Monday July 22, 2019 - Small culture

Tuesday July 23, 2019 - Miniprep & transformation

Split-NL

The Liu Addgene plasmid was purified from the overnight culture by an alternative "miniprep" protocol (due to the Qiagen kit being out of stock for the moment), giving the below peculiar concentrations:

	concentration (ng/μL)
Liu1a	1808.2
Liu2a	13.5
Liu3a	1415.8
Liu4a	518.7

We diluted Liu4a sample 20x and transformed it in both BL21 and Rosetta strains to test the expression of this dCas9 later on. We decided not to use these samples for PCR, first awaiting the expression results and hopefully we can soon redo the miniprep to obtain more reliably isolated DNA.

Wednesday July 24, 2019 - Small culture

Thursday July 25, 2019 - PCR, digestion & expression

Split-NL

The Mach1 small culture was miniprepped, obtaining below plasmid concentrations:

	concentration (ng/μL)
mpLiu1	131.9
mpLiu2	192.9
mpLiu3	136.4
mpLiu4	124.2

Sample 4 was diluted 20x and used for overhang PCR to extract the dCas9 coding sequence including the relevant restriction sites for ligating it in our plasmid. A sample of the PCR mixture was analysed on agarose gel, showing a band at the expected length. The remainder of the mixture was cleaned-up using the Qiagen PCR purification kit. Final concentration: 85.5 ng/μL.

Our original Lbit and Sbit vectors were also digested to remove dCas9 and allow for insertion of the new dCas9 sequence.

The Liu Addgene construct was expressed (induced by 0.2 mM IPTG) in BL21 and Rosetta in LB at 18 °C, 180 rpm.

BRET

The original Lbit vector was digested with XhoI and NcoI to replace the full original construct and allow for insertion of the 3 BRET gBlocks. Together with the Split-NL digested vectors, this was purified by agarose gel, with subsequent gel extraction Qiagen kit.

Friday July 26, 2019 - Centrifugation, lysis, SDS-page,...

Monday July 29, 2019 - DNA struggles

August

Monday August 5, 2019 - More DNA struggles, success for Split-NL

Tuesday August 13, 2019 - Reorganization

Wednesday August 14, 2019 - Digestion, PCR amplification

Thursday August 15, 2019 - Meeting with supervisors

Friday August 16, 2019 - Gibson Assembly primers design

Monday August 19, 2019 - Transformation & ligation

Tuesday August 20, 2019 - Small culture & digestion

Wednesay August 21, 2019 - Large culture, ligation & digestion

Thursday August 22, 2019 - Purification, PCR

Friday August 23, 2019 - SDS & Bioluminescence test

Monday August 26, 2019 - Transformation

Tuesday August 27, 2019 - Sequencing & purification

Wednesday August 28, 2019 - Small cultures & SDS gel

Thursday August 29, 2019 - Expression & Q-Tof sample

Friday August 30, 2019 - Purification

September

Monday September 2, 2019 - Testing

Tuesday September 3, 2019 - Testing

Split-NL

We did more testing in triplo with 50 μL samples in a 384-well plate:

Only PBS + 0.1% BSA
10 nM SmallBit
10 nM LargeBit
10 nM SmallBit + 10 nM LargeBit
10 nM SmallBit + 10 nM LargeBit + 1.2nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM unspecific DNA
10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 0.6 nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 2.4 nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 0.6 nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 1.2 nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 2.4 nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 0.6 nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 1.2 nM DNA 2
10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 2.4 nM DNA 2

NanoGlo in 2000x dilution was added to each sample before testing.

In the afternoon, we tested with lower sensor concentrations in duplo:

Only PBS + 0.1% BSA
1 nM SmallBit + 1 nM LargeBit
0.5 nM SmallBit + 0.5 nM LargeBit
1 nM SmallBit + 1 nM LargeBit + 1.2 nM DNA 2
0.5 nM SmallBit + 0.5 nM LargeBit + 1.2 nM DNA 2
1 nM SmallBit + 1 nM LargeBit + sgRNA 2 + 1.2 nM unspecific DNA
0.5 nM SmallBit + 0.5 nM LargeBit + sgRNA 2 + 1.2 nM unspecific DNA
1 nM SmallBit + 1 nM LargeBit + sgRNA 2 + 1.2nM DNA 2
0.5 nM SmallBit + 0.5 nM LargeBit + sgRNA 2 + 1.2nM DNA 2

NanoGlo in 2000x dilution was added to each sample before testing.

When analyzing the results, the control samples 4 and 5 in test 1 and control samples 3, 4 and 5 in test 2 gave a very high signal. The signal was much higher than the samples containing sgRNA and target DNA. Therefore, we put samples on the SDS gel. However, we used a relatively high concentration of BSA in the buffer, which can clearly be seen on gel due to a band at the height of 50 kDa. Some smears can be observed around 300 kDa, indicating aggregation, but it cannot be seen clearly due to the presence of BSA.

Peking

The DNA from the piece of gel which was cut out yesterday was extracted. Nluc and Cluc were each separately ligated into the vector containing dCas9. After ligation, the DNA was transformed into NovaBlue.

Wednesay September 4, 2019 - Colony PCR

Tuesday September 5, 2019 - Testing

Split-NL

We read that dCas9 shows aggregation when incubated at 37 °C, and that this can be prevented by incubation at room temperature for at least 10 minutes. Therefore, we tested the sensor activity under different incubation conditions. Tests in duplo:

10 nM Sbit + 10 nM Lbit | incubated for 10 minutes at room temperature
10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | incubated for 10 minutes at room temperature
10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | incubated for 10 minutes at room temperature
10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | incubated for 10 minutes at room temperature
10 nM Sbit + 10 nM Lbit | incubated for 30 minutes at room temperature
10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | incubated for 30 minutes at room temperature
10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | incubated for 30 minutes at room temperature
10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | incubated for 30 minutes at room temperature
10 nM Sbit + 10 nM Lbit | incubated for 30 minutes at 37 °C
10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | incubated for 30 minutes at 37 °C
10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | incubated for 30 minutes at 37 °C
10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | incubated for 30 minutes at 37 °C

NanoGlo in 2000x dilution was added to each sample.

BRET

The sequencing for the BRET sensor showed a completely wrong sequence for all samples. However, when we further analyzed the sequence it completely aligns with the sequence for our Split-NL sensor. When our original vector was digested using the correct restriction enzymes, the bands on the gel were difficult to distinguish from each other. Probably both our original insert (of the Split-NL sensor) and the vector were present in the sliced gel. Therefore, the original insert was ligated into the vector. We tested both sample 10 and 11 for luminescence and BRET sample 10 showed a big peak, indicating the presence of LargeBit. BRET sample 11 did not show any luminescence.

Peking

Small cultures were miniprepped and transformed into BL21(DE3).

Monday September 9, 2019 - Small culture & Gibson

Tuesday September 10, 2019 - Sequencing samples & testing

Split-NL

Sequencing

Sequencing samples were made and sent to BaseClear:

Sample 1: Sbit 6 + dCas9-G247-FOR
Sample 2: Sbit 6 + dCas9-G485-FOR
Sample 3: Sbit 6 + dCas9-G717-FOR
Sample 4: Sbit 6 + dCas9-A932-FOR
Sample 5: Sbit 6 + dCas9-T1167-FOR
Sample 6: Lbit 13 + dCas9-G247-FOR
Sample 7: Lbit 13 + dCas9-G485-FOR
Sample 8: Lbit 13 + dCas9-G717-FOR
Sample 9: Lbit 13 + dCas9-A932-FOR
Sample 10: Lbit 13 + dCas9-T1167-FOR

testing

A test was performed where the interspace distance was varied. Incubation was done for 30 minutes at room temperature. Order of addition in the plate: (1) DNA (2) Sbit (3) Lbit. NanoGlo was added in a 2000x dilution. Samples tested:

10 nM Sbit + 10 nM Lbit
10 nM Sbit + 10 nM Lbit + 1.2 nM DNA 2
10 nM Sbit + 10 nM Lbit + 1.2 nM non-target DNA
10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 1 (interspace distance 15)
10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nMDNA 2 (interspace distance 17)
10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nMDNA 2 (interspace distance 20)
10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 2 (interspace distance 22)
10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25)
10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nM DNA 3 (interspace distance 27)
10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 3 (interspace distance 30)

BRET

Miniprepping of BRET no.3 & BRET no.8 (small cultures) was done. These are 2 colonies with the correct vector length during colony PCR. Afterwards, sequencing samples were made of these BRET samples and sent to BaseClear:

Sample 11: BRET 10 + T7.FOR
Sample 12: BRET 10 + dCas9-S1338-FOR
Sample 13: BRET 10 + dCas9-G247-FOR
Sample 14: BRET 10 + dCas9-G485-FOR
Sample 15: BRET 10 + dCas9-G717-FOR
Sample 16: BRET 10 + dCas9-A932-FOR
Sample 17: BRET 3 + T7.FOR
Sample 18: BRET 8 + T7.FOR

No colonies were present after transformation, so Gibson Assembly failed. When looking at the gel, this is not suprising.

Peking

The small cultures were added to the large cultures. At the end of the day the cultures were induced with IPTG to activate protein expression. Furthermore, new LB agar plates were made.

Wednesay September 11, 2019 - Overhang PCR

Tuesday September 12, 2019 - Testing & Transformation

Monday September 16, 2019 - Ligation & transformation

Tuesday September 17, 2019 - Ligation & transformation

Wednesday September 18, 2019 - Small cultures, digestion, ligation & transformation

Thursday September 19, 2019 - Testing & expression

Split-NL

It would be possible that the crRNA + tracrRNA is not working. Therefore, we decided to order complete sgRNA with IDT. Today we tested with the new sgRNA. The samples were incubated for 30 minutes at 37 °C and sgRNA was added in a 6x molar excess. NanoGlo was added in a 2000x dilution.

2 nM Sbit + 2 nM Lbit | 5mM DTT present in PBS
2 nM Sbit + 2 nM Lbit + 1.2 nM target DNA | 5 mM DTT present in PBS
2 nM Sbit + 2 nM Lbit + sgRNA + non-target DNA |5 mM DTT present in PBS
2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 10 |5 mM DTT present in PBS
2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 17 | 5 mM DTT present in PBS
2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 25 | 5 mM DTT present in PBS
2 nM Sbit + 2 nM Lbit | no DTT
2 nM Sbit + 2 nM Lbit + 1.2 nM target DNA | no DTT
2 nM Sbit + 2 nM Lbit + sgRNA + non-target DNA | no DTT
2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 10 | no DTT
2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 17 | no DTT
2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 25 | no DTT

Native-PAGE to determine if dCas9 binds to the target sequence.

Native PAGE sample and running buffer were prepared. Samples were made, many controls were included (see below). Samples were loaded on the gel and afterwards the gel was stained with SYBRGold to stain the DNA. Furthermore, the gel was stained with Coomassie overnight.

BRET

No colonies were present, indicating that ligation had failed once again. The ligation of part 2 was repeated and the ligation mixtures of yesterday were transformed again in NovaBlue cells. In addition, colony PCR of the single colony on the plate of 17/09 was performed. The positive control showed the correct band on the gel, but the colony did not.

Characterization

CT52-mNeonGreen

Two 1L large cultures were made (1 for each small culture) using kanamycin and LB medium. Small cultures were added to the large cultures and incubated at 37 °C and 160 rpm until OD600 = 0.6-0.7. Expression was induced at OD600 = 0.6 by adding IPTG.

NanoLuc

Transformation was not successful. The transformation of BRET ligation was not successful as well, so it would be possible that something went wrong during the transformation experiment. Therefore, transformation was performed again, in BL21.

Friday September 20, 2019 - Purification

Monday September 23, 2019 - Colony PCR & testing

Tuesday September 24, 2019 - Small cultures

Wednesday September 25, 2019 - Testing

Split-NL

We found out that we miscalculated the DNA concentration, so we have been using 6 pM DNA instead of 6 nM DNA. Therefore, we have only been measuring background signal. New tests were performed using the correct DNA amounts. 30 μL samples were created with a 2 nM protein concentration, a 6x molar excess of sgRNA and a 6 nM DNA concentration. The protein with sgRNA was incubated at 37 °C for 10 minutes and incubation of the complex with DNA was done for 30 minutes at room temperature. For half of the samples the sgRNA was incubated at 50 °C for 5 minutes before use, which could avoid secondary structure formation of the sgRNA before incubation with dCas9.

2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) | sgRNA incubation at 37 °C
2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) | sgRNA incubation at 37 °C
2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) | sgRNA incubation at 37 °C
2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | sgRNA incubation at 37 °C
2 nM Sbit + 2 nM Lbit + sgRNA 1 | sgRNA incubation at 37 °C
2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA
2 nM Sbit + 2 nM Lbit
2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) | sgRNA incubation at 50 °C
2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) | sgRNA incubation at 50 °C
2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) | sgRNA incubation at 50 °C
2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | sgRNA incubation at 50 °C
2 nM Sbit + 2 nM Lbit + sgRNA 1 | sgRNA incubation at 50 °C
2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA
2 nM Sbit + 2 nM Lbit
2 nM Sbit + 2 nM Lbit + sgRNA 2A + sgRNA 1B + target DNA | sgRNA incubation at 50 °C

BRET

After extracting the digested parts and backbones from yesterday's agarose gel using the Qiagen gel extraction kit , we tried two more single insert ligations for creating our BRET sensor construct:

/NheI/-BRETp3 -/XhoI/ in /XhoI/-pET28a(+) -/NheI/
/SacI/-BRETp2 -/NheI/ in /NheI/-pET28a(+) -/SacI/

Both were performed using T4 DNA ligase, in 20 and 25 μL reactions for BRETp2 and BRETp3 respectively and 50 ng backbone was used.

Thursday September 26, 2019 - Testing

Monday September 30, 2019 - Testing

October

Tuesday October 1, 2019 - Small cultures

Wednesday October 2, 2019 - Expression & testing

Thursday October 3, 2019 - Testing & purification

Friday October 4, 2019 - PCR for Gibson & Testing

Monday October 7, 2019 - Gibson Assembly & QAMH

Tuesday October 8, 2019 - Gibson Assembly, testing & QAMH

Wednesday October 9, 2019 - Gibson Assembly, testing & QAMH

Split-NL

To improve the signal-to-noise ratio, we decided to test with higher protein concentrations. We used interspace distance 30 for testing, since this gave the highest signal in previous tests. We under the same conditions as the previous tests, but we used the protein concentrations 2, 5 and 10 nM.

BRET

No colonies were present on all the plates. The agarose gel of the gBlocks which were PCRed showed unexpected results for part 2. The weight was lower as expected. The PCR reaction was tried again. However, the same results came out of the gel.

Phage experiments at QAMH Brussels

Luckily we did not throw away our seemingly failed cultures yesterday, but kept them in the shaking incubator instead, as this morning the media of one of the flasks was all turbid! OD₆₀₀ measurement confirmed the presence of a high bacterial cell density, with an OD₆₀₀ of 1.525. The other culture had obtained an OD₆₀₀ of 0.081 and appeared slightly turbid to the naked eye, hence it was put back in the incubator. At the end of the day this culture had reached an OD₆₀₀ of 0.604. To check the linearity of the OD₆₀₀ vs. cell concentration we prepared a serial two-fold dilution series and measured the OD₆₀₀ giving the below, nicely linear, results:

Dilution	OD₆₀₀ culture 1	OD₆₀₀ culture 2
1x	1.475	0.601
2x	0.825	0.304
4x	0.433	0.159
8x	0.224	0.075
16x	0.114	0.040
32x	0.057	0.019
20x	0.090

Figure 3: linearity of the OD₆₀₀ vs. cell concentration of culture 1 with R² = 0.9934 and culture 2 with R² = 0.9997.

We then started the bacterial growth rate determination experiment according to the protocol. Eight 70 mL cultures were used, being 20x dilutions of the culture described above, resulting in a starting OD₆₀₀ of 0.13 (measured in flask 8 just before start of the actual experiment). The 8 cultures differed in starting concentration of glucose:

0.0 g/L Glucose
0.01 g/L Glucose
0.25 g/L Glucose
0.5 g/L Glucose
1.0 g/L Glucose
2.0 g/L Glucose
4.0 g/L Glucose
8.0 g/L Glucose

A 1 mL sample was taken every 20 minutes to measure the OD₆₀₀ of each individual culture. At the same time, another 1 mL sample was stored in an Eppendorf tube and quickly frozen using liquid nitrogen to be analyzed for remaining glucose concentration later on. A set of calibration samples was also prepared covering the starting glucose concentrations of the experimental cultures and included E. coli at an OD₆₀₀ of 0.163.

Thursday October 10, 2019 - Transformation, testing & QAMH

Friday October 11, 2019 - Testing

Split-NL

We tested the limit of detection of our sensor using 2 nM and 10 nM protein. A range of DNA concentrations from 1 pM to 10 nM was tested. The incubation conditions were the same as in previous tests and NanoGlo was added in 2000x dilution.

BRET

Colonies were present one of the Gibson assembly samples, the control for Gibson and the control for transformation! The colonies from Gibson were very small at first, so they were put back into the stove and taken out at the end of the day.

Phage experiments at QAMH Brussels

This morning we found our very first phage plaques! The plaques of yesterday's 24 plates were counted, but unfortunately no clear decline in unadsorbed phage could be seen over the 10 minutes sampling time. Therefore, we decided to plate the samples we stored in the fridge again today, to see if the unexpected result might be due to plating errors. Only the samples from the 10^-6 dilution were plated again, and using 200μL instead of 100μL this time to increase the number of plaques, aiding counting. We also plated a 10-fold serial dilution series of the T7 phage stock in order to re-titer the stock. Later the same day we could already count the plaques, but unfortunately we found the same overall result for the adsorption rate experiment. For the stock we found:

Dilution	Plaques	PFU/ml
10^-7	518	5E⁺¹⁰
10^-7	68	7E⁺¹⁰
10^-7	9	9E⁺¹⁰
Average		7E⁺¹⁰

The glucose concentrations of the samples of 9 October were also measured using the Hitachi Roche cobas 6000. The results showed some dubious deviations from the expected curve, but we will still try to use it to fit our model.

We also performed the infection assay experiment, first growing a diluted overnight culture, starting from OD₆₀₀ 0.104, reaching 0.126 when starting the actual experiment. To a 66 mL culture, 7*10⁶PFU/ml phage was added, resulting in a 7*10⁴PFU/ml solution. 3.8 ml growth medium and 15 droplets of chloroform were added in multiple falcon tubes to collect samples of 0.2 ml cell/phage suspension in. Moreover, 1 ml samples were collected in cuvettes for OD₆₀₀ measurements as well as 1 ml samples for glucose concentration measurements. The glucose samples will be analyzed next week, while part of the chloroformed samples were already plated (plaque assay) in various dilutions. The remainder will be plated next week.

Monday October 14, 2019 - Testing, small cultures & miniprepping

Tuesday October 15, 2019 - Testing & PCR

Split-NL

We wanted to make a calibration curve using NanoLuc-mNeonGreen. We tested a range of DNA concentrations using just our sensor without NanoLuc-mNeonGreen and we tested the same range of DNA concentrations with the addition of NanoLuc-mNeonGreen.

BRET

To confirm the negative colony PCR, a regular PCR reaction was performed to check the ligation of the gBlocks. T7 reverse and forward primers were also used the look at what is inside the vector.

Phage experiments at QAMH Brussels

The plaques of last Friday's plates were counted and the remaining samples that were not yet plated, were plated at several relevant dilutions. Later that day we could already count the plaques on the plates with a countable number of plaques. Unfortunately, not of all sampling times the titer could be determined, but with the available datapoints we found a clear overall pattern that matched our expectations.

Time (min)	Phage [PFU/ml]
0	7.00E⁺³
20	3.67E⁺⁰³
40	6.67E⁺⁰³
60	9.41E⁺⁰⁴
80	9.99E⁺⁰⁵
120	1.80E⁺⁰⁷
160	4.60E⁺⁰⁷
180	4.00E⁺⁰⁷
200	7.00E⁺⁰⁷
220	3.49E⁺⁰⁸

Figure 5: Phage growth plotted over time.

Griet Steurs helped us out by starting a bacterial culture from the plated DSM 613 for us on Sunday already. This culture was subcultured and grown for use in a repetition of the adsorption rate determination experiment. Unfortunately the bacteria appeared to grow only very slowly, so we didn't perform the experiment and instead started some additional liquid cultures for use tomorrow.

We also heat denatured (70°C, 2 hours) a 10-fold serial dilution (from 10⁰ to 10^-9) of the phage stock for experiments in Eindhoven, to try to detect the freed phage DNA.

The glucose measurement samples collected last Friday during the infection assay were also measured using the Roche Hitachi cobas 6000.

Wednesday October 16, 2019 - Testing

Thursday October 17, 2019 - Testing

Friday October 18, 2019 - Testing

Monday October 21, 2019 - Testing

Milestones

Throughout the competition, a lot of milestones were achieved inside and outside the lab. Below you can find the list of the most important milestones we achieved together with our team.

February 6, 2019 - Kick-off meeting

March 27, 2019 - Team complete

March 28, 2019 - Officially registered for iGEM

April 15, 2019 - First meeting with an expert, Chris Arts

April 23, 2019 - Reached the top 40 of the TU/e contest

April 24, 2019 - Arrival of the iGEM distribution kit

May 2, 2019 - First team bonding activity

May 13, 2019 - Offical iGEM TU Eindhoven 2019 design

May 20, 2019 - Reached the top 20 of the TU/e contest

May 22, 2019 - Netherlands Biotechnology Congress (NBC), Ede

May 28, 2019 - First meeting at Queen Astrid Military Hospital, Brussels, with Jean-Paul Pirnay

June 6, 2019 - 2^nd place at TU/e contest final event

June 11, 2019 - New office

June 13, 2019 - iGEM Europe meetup, The Hague

June 13, 2019 - First patient talk, Queen Astrid Military Hospital, Brussels

June 14, 2019 - Phage therapy symposium, Queen Astrid Military Hospital, Brussels

June 17, 2019 - First day in the lab, because of arrival vector

June 17, 2019 - Arrival team clothing and team photoshoot

July 11, 2019 - First sponsor: DSM

July 17, 2019 - Article about iGEM TU Eindhoven on FMT gezondheidszorg

August 17, 2019 - Dutch Meet-Up Leiden and Groningen

August 22, 2019 - Formation of our dCas9-Split-NanoLuc

August 26, 2019 - Crowdfunding online

August 26, 2019 - Official GO for the team

August 29, 2019 - Booking of tickets to Boston

September 2, 2019 - Team project name: dCastect

September 7, 2019 - Reaching 50% of our crowdfunding campaign

September 19, 2019 - Second team bonding evening

September 21, 2019 - Article in the Eindhovens Dagblad (ED)

September 21, 2019 - Reaching 100% of our crowdfunding campaign

September 25, 2019 - Positive test results of our dCas9-Split-NanoLuc

October 2, 2019 - Arrival Postcards Duesseldorf collaboration

October 7, 2019 - Performing our phage experiments at the QAMH in Brussels

October 22, 2019 - Successful Wiki Freeze!

October 25, 2019 - BeNeLux Mini Jamboree, Eindhoven

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                      More information about our achievements outside the lab is shown on the <a class="pagelink" onclick="changeTab('milestones', this)">Milestones</a> page.
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                                  <img src="https://2019.igem.org/wiki/images/9/97/T--TU_Eindhoven--Lab20190619.png" alt="Process" width="90%" />
-                                 </br>Fig. 1: Culture plates.
+                                 </br>Figure 1: Culture plates.
                              </div>
+							</br>
                          </div>
                      </div>
@@ Line 76: / Line 79: @@
                  <div class="box middle notebook" data-modal="modal4">
-                     Thursday June 20, 2019 - Miniprep and protein expression
+                     Thursday June 20, 2019 - Miniprep & protein expression
                  </div>
                  <div class="modal" id="modal4">
                      <div class="modal-content">
-                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday June 20, 2019 - Miniprep and protein expression</h2></div>
+                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday June 20, 2019 - Miniprep & protein expression</h2></div>
                          <div class="modal-body">
                              <p>
                                  The small cultures were taken out of the incubator. The two cultures with BL21(DE3) cells were put on ice for protein expression later in the morning.
                                  The plasmids from the other four cultures with NovaBlue cells were isolated using the QIAprep miniprep kit. The following DNA concentrations were obtained:
-.8 ng/μL, 225.0 ng/μL, 202.0 ng/μL, 215.2 ng/μL. The samples were put in the freezer (-20oC). Tomorrow these samples will be digested and ligated to form dCas9-Lbit.
+.8 ng/μL, 225.0 ng/μL, 202.0 ng/μL, 215.2 ng/μL. The samples were put in the freezer (-20 °C). Tomorrow these samples will be digested and ligated to form dCas9-Lbit.
                              </p>
                              <p>
@@ Line 91: / Line 94: @@
                                  protein expression was initiated by adding IPTG. The large cultures were incubated overnight.
                              </p>
-                            <div class="table-caption">Table 1: DNA concentrations after miniprep</div>
-                            <table class="parts-table" align="center">
-                                <tr>
-                                    <th></th>
-                                    <th>Concentration (ng/μL)</th>
-                                </tr>
-                                <tr>
-                                    <td>1.</td>
-                                    <td>215.8</td>
-                                </tr>
-                                <tr>
-                                    <td>2.</td>
-                                    <td>225.0</td>
-                                </tr>
-                                <tr>
-                                    <td>3.</td>
-                                    <td>202.0</td>
-                                </tr>
-                                <tr>
-                                    <td>4.</td>
-                                    <td>215.2</td>
-                                </tr>
-                            </table>
-                            </br>
                          </div>
                      </div>
@@ Line 121: / Line 100: @@
                  <div class="box middle notebook" data-modal="modal5">
-                     Friday June 21, 2019 - Digestion and protein pellets
+                     Friday June 21, 2019 - Digestion & protein pellets
                  </div>
                  <div class="modal" id="modal5">
                      <div class="modal-content">
-                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday June 21, 2019 - Digestion and protein pellets</h2></div>
+                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday June 21, 2019 - Digestion & protein pellets</h2></div>
                          <div class="modal-body">
                              <p>
-                                 The three large cultures (hopefully with the protein dCas9-Sbit inside the bacteria) were centrifuged in several rounds.
+                                 The three large cultures were centrifuged in several rounds.
                                  The volumes were too big for just one round. After each round, the supernatant was discarded. In the end the pellets were taken out of the centrifuge bottles
                                  and put into three falcon tubes (pellets from TB, 2YT and LB medium). The pellets were snap freezed and will be purified after the weekend.
@@ Line 136: / Line 115: @@
                                  to separate the digested part from the rest of the plasmid. The ladder had leaked into the samples, however we were still able to cut out the DNA of dCas9-Lbit.
                                  The DNA was retrieved from the agarose gel using the QIAquick Gel Extraction Kit. Due to lack of time, the sample will be ligated after the weekend.
                              </p>
-                            <div class="table-caption">Table 2: Nanodrop DNA concentrations after digestion</div>
-                            <table class="parts-table" align="center">
-                                <tr>
-                                    <th></th>
-                                    <th>Concentration (ng/μL)</th>
-                                    <th>260/280</th>
-                                    <th>260/230</th>
-                                </tr>
-                                <tr>
-                                    <td>1.</td>
-                                    <td>7.5</td>
-                                    <td>1.53</td>
-                                    <td>0.31</td>
-                                </tr>
-                                <tr>
-                                    <td>2.</td>
-                                    <td>9.3</td>
-                                    <td>1.76</td>
-                                    <td>0.44</td>
-                                </tr>
-                                <tr>
-                                    <td>3.</td>
-                                    <td>13.7</td>
-                                    <td>1.73</td>
-                                    <td>0.91</td>
-                                </tr>
-                            </table>
-                            </br>
                          </div>
                      </div>
@@ Line 171: / Line 122: @@
                  <div class="box middle notebook" data-modal="modal6">
-                     Monday June 24, 2019 - Ligation and column preparation
+                     Monday June 24, 2019 - Ligation & column preparation
                  </div>
                  <div class="modal" id="modal6">
                      <div class="modal-content">
-                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday June 24, 2019 - Ligation and column preparation</h2></div>
+                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday June 24, 2019 - Ligation & column preparation</h2></div>
                          <div class="modal-body">
                              <p>
@@ Line 183: / Line 134: @@
                              </p>
-                            <div class="table-caption">Table 3: Ligation mixtures (calculated based on DNA concentration</div>
-                            <table class="parts-table" align="center">
-                                <tr>
-                                    <th></th>
-                                    <th>50 ng vector (μL)</th>
-                                    <th>MilliQ (μL)</th>
-                                </tr>
-                                <tr>
-                                    <td>1.</td>
-                                    <td>6.67</td>
-                                    <td>10.33</td>
-                                </tr>
-                                <tr>
-                                    <td>2.</td>
-                                    <td>5.4</td>
-                                    <td>11.6</td>
-                                </tr>
-                                <tr>
-                                    <td>3.</td>
-                                    <td>3.65</td>
-                                    <td>13.35</td>
-                                </tr>
-                            </table>
-                            </br>
                          </div>
                      </div>
@@ Line 213: / Line 140: @@
                  <div class="box middle notebook" data-modal="modal7">
-                     Tuesday June 25, 2019 - Purification and small culture
+                     Tuesday June 25, 2019 - Purification & small culture
                  </div>
                  <div class="modal" id="modal7">
                      <div class="modal-content">
-                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday June 25, 2019 - Purification and small culture</h2></div>
+                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday June 25, 2019 - Purification & small culture</h2></div>
                          <div class="modal-body">
                              <p>
-                                 The pellets of dCas9-Sbit were taken out of the -80oC freezer and put on ice to thaw. Subsequently, the pellets were dissolved and lysed (chemically).
+                                 The pellets of dCas9-Sbit were taken out of the -80 °C freezer and put on ice to thaw. Subsequently, the pellets were dissolved and lysed (chemically).
                                  When the lysing was finished, the proteins were purified using <mark class="yellow">IMAC columns</mark>. The elution from the IMAC columns were then put on the <mark class="green">strep columns</mark>.
                                  Amicon filters were used to concentrate the elution from the strep columns. NanoDrop was used to measure if there were any proteins in the solution.
@@ Line 239: / Line 166: @@
                  <div class="box middle notebook" data-modal="modal8">
-                     Wednesday June 26, 2019 - Miniprep and SDS-PAGE
+                     Wednesday June 26, 2019 - Miniprep & SDS-PAGE
                  </div>
                  <div class="modal" id="modal8">
                      <div class="modal-content">
-                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday June 26, 2019 - Miniprep and SDS-PAGE</h2></div>
+                         <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday June 26, 2019 - Miniprep & SDS-PAGE</h2></div>
                          <div class="modal-body">
                              <p>
@@ Line 251: / Line 178: @@
                                  After this, the gel is washed in water overnight on the shaking table.
                              </p>
-                             <div class="table-caption">Table 4: DNA concentrations dCas9-Lbit</div>
-                            <table class="parts-table" align="center">
-                                <tr>
-                                    <th></th>
-                                    <th>Concentration (ng/μL)</th>
-                                    <th>260/280</th>
-                                    <th>260/230</th>
-                                </tr>
-                                <tr>
-                                    <td>1.</td>
-                                    <td>44</td>
-                                    <td>1.79</td>
-                                    <td>1.82</td>
-                                </tr>
-                                <tr>
-                                    <td>2.</td>
-                                    <td>180</td>
-                                    <td>1.84</td>
-                                    <td>1.97</td>
-                                </tr>
-                                <tr>
-                                    <td>3.</td>
-                                    <td>204.1</td>
-                                    <td>1.87</td>
-                                    <td>2.22</td>
-                                </tr>
-                                <tr>
-                                    <td>4.</td>
-                                    <td>194.4</td>
-                                    <td>1.87</td>
-                                    <td>2.13</td>
-                                </tr>
-                                <tr>
-                                    <td>5.</td>
-                                    <td>198.9</td>
-                                    <td>1.86</td>
-                                    <td>2.21</td>
-                                </tr>
-                                <tr>
-                                    <td>6.</td>
-                                    <td>209.5</td>
-                                    <td>1.89</td>
-                                    <td>2.28</td>
-                                </tr>
-                                <tr>
-                                    <td>7.</td>
-                                    <td>211.4</td>
-                                    <td>1.86</td>
-                                    <td>2.19</td>
-                                </tr>
-                                <tr>
-                                    <td>8.</td>
-                                    <td>190.5</td>
-                                    <td>1.87</td>
-                                    <td>2.18</td>
-                                </tr>
-                                <tr>
-                                    <td>9.</td>
-                                    <td>223.8</td>
-                                    <td>1.86</td>
-                                    <td>2.14</td>
-                                </tr>
-                                <tr>
-                                    <td>10.</td>
-                                    <td>209.9</td>
-                                    <td>1.85</td>
-                                    <td>2.19</td>
-                                </tr>
-                            </table>
-                            </br>
                          </div>
                      </div>
@@ Line 327: / Line 185: @@
              <div class="box middle notebook" data-modal="modal9">
-                 Thursday June 27, 2019 - SDS-PAGE + sequencing + new LB stock
+                 Thursday June 27, 2019 - SDS-PAGE, sequencing & new LB stock
              </div>
              <div class="modal" id="modal9">
                  <div class="modal-content">
-                     <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday June 27, 2019 - SDS-PAGE + sequencing + new LB stock</h2></div>
+                     <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday June 27, 2019 - SDS-PAGE, sequencing & new LB stock</h2></div>
                      <div class="modal-body">
                          <div class="photo-center">
                              <img src="https://2019.igem.org/wiki/images/4/49/T--TU_Eindhoven--Lab20190627.png" alt="Process" width="90%" />
-                             </br>Fig. 1: SDS-PAGE gels.
+                             </br>Figure 2: SDS-PAGE gels.
                          </div>
+						</br>
                      </div>
                  </div>
@@ Line 343: / Line 202: @@
              <div class="box middle notebook" data-modal="modal10">
-                 Friday June 28, 2019 - 2nd Transformation Sbit + IPTG stock
+                 Friday June 28, 2019 - 2<sup>nd</sup> Transformation Sbit, IPTG stock & New plan expression
              </div>
              <div class="modal" id="modal10">
                  <div class="modal-content">
-                     <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday June 28, 2019 - 2nd Transformation Sbit + IPTG stock</h2></div>
+                     <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday June 28, 2019 - 2<sup>nd</sup> Transformation Sbit, IPTG stock & New plan expression</h2></div>
                      <div class="modal-body">
                          <p>
@@ Line 364: / Line 223: @@
                              Hence, for 1mM final concentration, add <b>1.2 ml stock IPTG to 1L medium</b>. For 200nM final concentration, add <b>0.25 ml stock IPTG</b>.
                          </p>
+						<h3>New plan Expression</h3>
-                    </div>
+						<p>
-                </div>
-            </div>
-            <div class="box middle notebook" data-modal="modal11">
-                Friday June 28, 2019 - New plan expression
-            </div>
-            <div class="modal" id="modal11">
-                <div class="modal-content">
-                    <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday June 28, 2019 - New plan expression</h2></div>
-                    <div class="modal-body">
-                        <p>
                              From the SDS-PAGE results we concluded that no dCas9-Sbit was formed. Therefore a new plan was made to test different combinations of expression conditions based on
                              information we gathered. The plan is to test four different condition combinations.
@@ Line 383: / Line 230: @@
                          <p>
                              The following conditions will be the same:
-                             <ul>
+                             <ul class="list">
                                  <li>Host: BL21(DE3)</li>
                                  <li>Medium: 750 mL LB</li>
@@ Line 390: / Line 237: @@
                              </ul>
                          </p>
-                        <div class="table-caption">Table 5: Further conidition combinations</div>
-                        <table class="parts-table" align="center">
-                            <tr>
-                                <th></th>
-                                <th>IPTG (mM)</th>
-                                <th>Temperature (°C)</th>
-                            </tr>
-                            <tr>
-                                <td>1.</td>
-                                <td>0.2</td>
-                                <td>16</td>
-                            </tr>
-                            <tr>
-                                <td>2.</td>
-                                <td>1.0</td>
-                                <td>16</td>
-                            </tr>
-                            <tr>
-                                <td>3.</td>
-                                <td>0.2</td>
-                                <td>20</td>
-                            </tr>
-                            <tr>
-                                <td>4.</td>
-                                <td>1.0</td>
-                                <td>20</td>
-                            </tr>
-                        </table>
-                        </br>
                      </div>
                  </div>
              </div>
+			</br>
+			<h3>July</h3>
+			<div class="box middle notebook" data-modal="modal11">
+                    Monday July 1, 2019 - Small culture
+                </div>
+                <div class="modal" id="modal11">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday July 1, 2019 - Small culture</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                Two small 8 mL LB liquid cultures (incl. 8 μL Kanamycin)of two of the dCas9-NL-Sbit transformants were started for growing overnight, to act as starter
+								cultures for protein expression tomorrow. Two large erlenmeyer flasks with 750 mL LB medium were prepared and placed in the autoclave queue for expression
+								tomorrow.
+                            </p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal12">
+                    Tuesday July 2, 2019 - Small culture
+                </div>
+                <div class="modal" id="modal12">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday July 2, 2019 - Small culture</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                Since the large culture media couldn't be autoclaved before the end of today, we decided to postpone expression one day and to prepare a fresh small culture.
+								Hence, another two small 8 mL LB liquid cultures (incl. 8 μL Kanamycin)of two of the remaining dCas9-NL-Sbit transformants were started for growing overnight,
+								to act as starter culture for protein expression tomorrow.
+                            </p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal13">
+                    Wednesday July 3, 2019 - Expression
+                </div>
+                <div class="modal" id="modal13">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday June 28, 2019 - Expression</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                As the initial attempt of expressing the dCas9-NL-Sbit fusion protein failed, we now try 2 sets of different expression conditions in parallel (we narrowed
+								down to only 2 condition sets, in contrast to the 4 we planned to do initially to save work and time, while still using the most interesting conditions).
+								Namely:
+                            </p>
+							<table class="parts-table" align="center">
+								<tr>
+									<th></th>
+									<th>[IPTG]</th>
+									<th>Temperature (°C)</th>
+									<th>Color label</th>
+								</tr>
+								<tr>
+									<th>1.</th>
+									<th>0.2 mM</th>
+									<th>16</th>
+									<th>Green</th>
+								</tr>
+								<tr>
+									<th>2.</th>
+									<th>1 mM</th>
+									<th>16</th>
+									<th>Blue</th>
+								</tr>
+							</table>
+							<p>
+								Both cultures were at 750 mL scale, with 0.75 mL 1000x IPTG added. The cultures were grown at 37 °C, 160 rpm until reaching OD<sub>600</sub> ~0.5-0.6, upon
+								which IPTG was added according to the scheme below.
+							</p>
+							<table class="parts-table" align="center">
+								<tr>
+									<th></th>
+									<th>Color label</th>
+									<th>[IPTG]</th>
+									<th>Add this volume of 0.84M IPTG stock solution to 750 ml medium</th>
+								</tr>
+								<tr>
+									<th>1.</th>
+									<th>Green</th>
+									<th>0.2 mM</th>
+									<th>0.18 ml</th>
+								</tr>
+								<tr>
+									<th>2.</th>
+									<th>Blue</th>
+									<th>1 mM</th>
+									<th>0.90 ml</th>
+								</tr>
+							</table>
+							<p>
+								The cultures were the incubated overnight at 16°C, 160 rpm.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal14">
+                    Thursday July 4, 2019 - Centrifugation
+                </div>
+                <div class="modal" id="modal14">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday July 4, 2019 - Centrifugation</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                The overnight Sbit expression cultures were distributed over two 500 mL ultracentrifuge tubes each and spun down at 8600 x g for 15 + 10 minutes at 4 °C.
+								The resulting pellets were dissolved with 5 mL MQ H<sub>2</sub>O each. This was then added together per culture (keeping the two different cultures apart) in
+								a Falcon tube and centrifuged for 10 minutes at 3000 rpm at 4 °C. The pellet was then stored at -80 °C for lysis and protein purification to be performed
+								tomorrow. We also received instructions on how to use the ultrasound sonication cell lysis device for use tomorrow. We also received instructions on how
+								to use the ultrasound sonication cell lysis device for use tomorrow. We also prepared the basis of a sonication lysis buffer (50 mM Tris, pH 8.0, 500 mM NaCl).
+                            </p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal15">
+                    Friday July 5, 2019 - Sonication lysis & protein purification
+                </div>
+                <div class="modal" id="modal15">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday July 5, 2019 - Sonication lysis & protein purification</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                Yesterday's cell pellets, hopefully containing dCas9-NL-Sbit, were thawed on ice and resuspended in sonication lysis buffer, resulting in about 25 mL total
+								volume, to which a protease inhibitor mix tablet (Merck, without EDTA!) was added, as well as lysozyme. These suspensions were then lysed by sonication
+								according to the sonication protocol using a Branson 150 sonifier.
+                            </p>
+							<p>
+								The resulting lysates were then centrifuged at 20k g for 30 min at 4 °C and the resulting supernatants were then added onto separate washed NiNTA columns for
+								His-tag affinity purification. Afterwards, the eluates were further purified using strep tag columns. Nanodrop measurement of the resulting eluates (using
+								protein A280 setting, with exctinction coefficient and MW obtained through the ExPASy online tool) unfortunately indicated that basically no dCas9-Sbit
+								protein was present. We then also put the flow through from the His purification over the Strep columns, to test for presence of protein that might not have
+								bound tightly to the NiNTA column. Unfortunately, also here the Nanodrop determined concentrations were very low.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal16">
+                    Monday July 8, 2019 - SDS-PAGE
+                </div>
+                <div class="modal" id="modal16">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday July 8, 2019 - SDS-PAGE</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                An SDS-PAGE gel was prepared for all purification fractions of both cultures of last week. We also had a meeting with some of our supervisors to discuss the
+								expression problem we are facing as well as our general progress. Moreover, our new idea for an additional interesting new dCas9 based dsDNA sensor part was
+								presented.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal17">
+                    Tuesday July 9, 2019 - SDS-PAGE
+                </div>
+                <div class="modal" id="modal17">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday July 9, 2019 - SDS-PAGE</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                Yesterday's gels were photographed and analyzed. We would expect a band at the MW, namely 164 kDa, to indicate the presence of our dCas9-Sbit fusion protein.
+								We do see very dim bands in both the pellet and His flow through fractions around this height, but not in the elute fractions were they would be expected.
+							</p>
+							<h3>Peking constructs</h3>
+							<p>
+								We designed and ordered split Firefly luciferase DNA constructs from Twist Bioscience to be able to express the dCas9 split-luciferase sensor designed by iGEM Peking 2015 (not available from the Bio Brick repository) to compare it to ours.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal18">
+                    Wednesday July 10, 2019 - Denaturing His-tag purification
+                </div>
+                <div class="modal" id="modal18">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday July 10, 2019 - Denaturing His-tag purification</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                We performed another round of His-tag purification with fractions from the previous purification rounds, now under denaturing conditions, namely with 8M
+								urea added. This in order to test the hypothesis that the His-tag might be unavailable for binding the NiNTA column in the protein's native condition. We
+								only saw a very dim band, so we think this is a rather unlikely explanation, and that the problem rather lies in codon related issues. Still, we'd like to
+								test this hypothesis. The resulting fractions were put on an SDS-PAGE gel, which was washed overnight.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal19">
+                    Thursday July 11, 2019 - Denaturation
+                </div>
+                <div class="modal" id="modal19">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday July 11, 2019 - Denaturation</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+                                The gel made yesterday doesn't appear to support the unavailable His-tag hypothesis, hence we will need to troubleshoot in a different direction. We discussed
+								the situation with supervisors and concluded the problem most likely lies in the alternative codons we used in our expression plasmid (which was codon optimized
+								for E. coli, which frankly appears to work counteractive for expression). We therefore decided to order a dCas9 (wild type codons) containing plasmid from
+								Addgene (Plasmid #62935)to extract this sequence by overhang PCR and replace the dCas9 sequence in our own construct by restriction/ligation.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								In the meantime, we started designing the DNA construct for our dCas9-NanoLuc BRET sensor part. We decided, taking the large size of the dCas9 coding sequence
+								into account, that it would be a good and economical idea to orderthe construct as 3 gBlocks from IDT, to be ligated to each other and into our pET-28a(+)
+								expression plasmid.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal20">
+                    Friday July 12, 2019 - Design
+                </div>
+                <div class="modal" id="modal20">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday July 12, 2019 - Design</h2></div>
+                        <div class="modal-body">
+                            <p>
+								We worked on the design of the BRET sensor construct, the test target DNA needed for future testing using both sensors and ordered the selected dCas9 construct from Addgene.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal21">
+                    Monday July 15, 2019 - General
+                </div>
+                <div class="modal" id="modal21">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday July 15, 2019 - General</h2></div>
+                        <div class="modal-body">
+                            <h3>July 15 - 19, 2019</h3>
+							<p>
+								We continued working on the design of the DNA construct for our BRET sensor idea this week, as well as on the synthetic test target dsDNA strands and crRNA.
+								Moreover, protocols were prepared for cloning the dCas9 sequence from the Liu Addgene construct into our vector.
+							</p>
+							<p>
+								On Friday, the designed BRET constructs (3 gBlocks) were ordered at IDT, as well as primers for overhang PCR of the Liu Addgene construct. The Addgene order
+								was delivered and the bacterial stab was plated for growing over the weekend.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal22">
+                    Monday July 22, 2019 - Small culture
+                </div>
+                <div class="modal" id="modal22">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday July 22, 2019 - Small culture</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+								A Mach1 bacterial colony from the plate grown over the weekend containing the Liu plasmid obtained through Addgene was cultured in 5 mL LB with 5 μl Ampicillin
+								overnight. Moreover, some additional stocks were prepared, like ampicillin and chloramphenicol (for use with Rosetta strain cells) stocks. Also LB-Agar plates
+								were poured, with different antibiotics (Kanamycin, Ampicillin, Chloramphenicol and combinations).
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal23">
+                    Tuesday July 23, 2019 - Miniprep & transformation
+                </div>
+                <div class="modal" id="modal23">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday July 23, 2019 - Miniprep & transformation</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+								The Liu Addgene plasmid was purified from the overnight culture by an alternative "miniprep" protocol (due to the Qiagen kit being
+								out of stock for the moment), giving the below peculiar concentrations:
+							</p>
+							<table class="parts-table" align="center">
+								<tr>
+									<th></th>
+									<th>concentration (ng/μL)</th>
+								</tr>
+								<tr>
+									<th>Liu1a</th>
+									<th>1808.2</th>
+								</tr>
+								<tr>
+									<th>Liu2a</th>
+									<th>13.5</th>
+								</tr>
+								<tr>
+									<th>Liu3a</th>
+									<th>1415.8</th>
+								</tr>
+								<tr>
+									<th>Liu4a</th>
+									<th>518.7</th>
+								</tr>
+							</table>
+							<p>
+								We diluted Liu4a sample 20x and transformed it in both BL21 and Rosetta strains to test the expression of this dCas9 later on. We decided not to use these
+								samples for PCR, first awaiting the expression results and hopefully we can soon redo the miniprep to obtain more reliably isolated DNA.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal24">
+                    Wednesday July 24, 2019 - Small culture
+                </div>
+                <div class="modal" id="modal24">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday July 24, 2019 - Small culture</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+								Transformations in BL21 and Rosetta appeared successful. Small cultures of the Liu Addgene plasmid in BL21, Rosetta and the original carrier strain (Mach1)
+								were prepared (5 mL LB + Amp for Mach1, 8 mL LB + Amp for BL21 and 8 mL LB + Amp + Cam for Rosetta).
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal25">
+                    Thursday July 25, 2019 - PCR, digestion & expression
+                </div>
+                <div class="modal" id="modal25">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday July 25, 2019 - PCR, digestion & expression</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+								The Mach1 small culture was miniprepped, obtaining below plasmid concentrations:
+							</p>
+							<table class="parts-table" align="center">
+								<tr>
+									<th></th>
+									<th>concentration (ng/μL)</th>
+								</tr>
+								<tr>
+									<th>mpLiu1</th>
+									<th>131.9</th>
+								</tr>
+								<tr>
+									<th>mpLiu2</th>
+									<th>192.9</th>
+								</tr>
+								<tr>
+									<th>mpLiu3</th>
+									<th>136.4</th>
+								</tr>
+								<tr>
+									<th>mpLiu4</th>
+									<th>124.2</th>
+								</tr>
+							</table>
+							<p>
+								Sample 4 was diluted 20x and used for overhang PCR to extract the dCas9 coding sequence including the relevant restriction sites for ligating it in our plasmid.
+								A sample of the PCR mixture was analysed on agarose gel, showing a band at the expected length. The remainder of the mixture was cleaned-up using the Qiagen PCR
+								purification kit. Final concentration: 85.5 ng/μL.
+							</p>
+							<p>
+								Our original Lbit and Sbit vectors were also digested to remove dCas9 and allow for insertion of the new dCas9 sequence.
+							</p>
+							<p>
+								The Liu Addgene construct was expressed (induced by 0.2 mM IPTG) in BL21 and Rosetta in LB at 18 °C, 180 rpm.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								The original Lbit vector was digested with XhoI and NcoI to replace the full original construct and allow for insertion of the 3 BRET gBlocks. Together with
+								the Split-NL digested vectors, this was purified by agarose gel, with subsequent gel extraction Qiagen kit.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal26">
+                    Friday July 26, 2019 - Centrifugation, lysis, SDS-page,...
              </div>
+                <div class="modal" id="modal26">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday July 26, 2019 - Centrifugation, lysis, SDS-page,...</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								The overnight expression cultures were pelleted, resuspended in sonication lysis buffer en lysed by sonication. Subsequently, the lysate was centrifuged and
+								SDS-PAGE samples were made of both the supernatant and pellet and run on a gel to check for clear presence of the dCas9 protein.
+							</p>
+							<p>
+								The PCRed insert was digested, as was the His-MBP gBlock to be coupled to the dCas9 in the plasmid.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								The 3 gBlocks were digested with the relevant restriction enzymes. However, we only have 1 μg of each, which would all be used in a single regular digestion
+								reaction! Therefore we tried with 0.1 μg and decided we would order primers and PCR amplify the gBlocks next week.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal27">
+                    Monday July 29, 2019 - DNA struggles
              </div>
+                <div class="modal" id="modal27">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday July 29, 2019 - DNA struggles</h2></div>
+                        <div class="modal-body">
+							<h3>July 29 - August 2</h3>
+							<h3>Split-NL</h3>
+							<p>
+								This week, we attempted to ligate the digested Liu Addgene dCas9 construct into our digested pET28a(+) vector together with the His-MBP tag gBlock.
+								Unfortunately, even after multiple attempts, this was unsuccessful. We discussed this with our lab supervisor and then decided to order a new primer to be
+								able to PCR the dCas9 sequence from the Addgene plasmid over again, now including a restriction site allowing us to insert the dCas9 into the vector directly,
+								without the need for the tag gBlock.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Similar issues were faced for the BRET sensor construct. Multiple attempts at ligating the 3 gBlocks to each other and into the vector, either all at once,
+								or sequentially, were all unsuccessful. Primers for amplifying the gBlocks were designed and ordered for use next week.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			</br>
+			<h3>August</h3>
+			<div class="box middle notebook" data-modal="modal99">
+                    Monday August 5, 2019 - More DNA struggles, success for Split-NL
+            </div>
+                <div class="modal" id="modal99">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday August 5, 2019 - More DNA struggles, success for Split-NL</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+                            <p>
+								Using the new primers, overhang PCR was again performed on the Liu Addgene plasmid to extract the dCas9 sequence. This was performed successfully, as indicated by agarose gel analysis. Subsequently, this DNA was digested with the relevant restriction enzymes and ligated into the correspondingly digested Sbit and Lbit vectors. Transformation of both ligation mixtures in NovaBlue resulted in colonies, which were screened for presence of the correct insert by colony PCR. The colony PCR was positive for most of the colonies and many of these were miniprepped, resulting in multiple DNA samples of both Liu-dCas9-Lbit and -Sbit. These were transformed in BL21 for subsequent expression later on.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								After successful PCR amplification of all gBlocks, again multiple attempts at ligating the 3 gBlocks to each other and into the vector, either all at once, or sequentially, were all unsuccessful. A ligation of only the two most relevant CP-dCas9 spanning gBlocks into a correspondingly digested backbone appeared successful at first based on colony PCR, but later on turned out to be unsuccessful as well. We will look into other options next week.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal100">
+                    Tuesday August 13, 2019 - Reorganization
+            </div>
+                <div class="modal" id="modal100">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday August 13, 2019 - Reorganization</h2></div>
+                        <div class="modal-body">
+                            <p>We reorganized the blue boxes containing all DNA, enzymes and reagents in the -20 °C freezer.</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal101">
+                    Wednesday August 14, 2019 - Digestion, PCR amplification
+                </div>
+                <div class="modal" id="modal101">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday August 14, 2019 - Digestion, PCR amplification</h2></div>
+                        <div class="modal-body">
+                            <h3>Split-NL</h3>
+							<p>
+								The expressed Liu Addgene construct was purified using IMAC. Afterwards, SDS-PAGE analysis was performed to check whether the correct protein
+								was retrieved. A band around 170 kDa was expected and the SDS-PAGE gel indeed shows a band just above 150 kDa.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Parts 2 and 3 were PCR amplified to retrieve enough DNA for digestion and ligation. Subsequently, both parts were digested using the restriction
+								site NheI. The digested parts were separated using an agarose gel and excised.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal102">
+                    Thursday August 15, 2019 - Meeting with supervisors
+            </div>
+                <div class="modal" id="modal102">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday August 15, 2019 - Meeting with supervisors</h2></div>
+                        <div class="modal-body">
+                            <h3>General</h3>
+							<p>
+								Today we had a meeting with our supervisors. We presented our progress and discussed about some problems we encountered in the lab.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								We performed gel extraction of BRET part 2 and BRET part 3 after NheI digestion. The DNA concentrations were measured using NanoDrop.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal103">
+                    Friday August 16, 2019 - Gibson Assembly primers design
+            </div>
+                <div class="modal" id="modal103">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday August 16, 2019 - Gibson Assembly primers design</h2></div>
+                        <div class="modal-body">
+							<h3>BRET</h3>
+                            <p>
+								For BRET it was decided that Gibson Assembly would be performed instead of regular digestion and ligation. The gBlocks we ordered cannot directly be used for Gibson,
+								since the gBlocks do not contain overlap. We designed overhang primers for the gBlocks to make them suitable for Gibson assembly and ordered them from IDT.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal104">
+                    Monday August 19, 2019 - Transformation & ligation
+            </div>
+                <div class="modal" id="modal104">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday August 19, 2019 - Transformation & ligation</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+                            <p>
+								After successful ligation of the Liu Addgene dCas9 sequence into our original vector, the plasmid was transformed into BL21 cells. We used four plates:
+for Sbit and 2 for Lbit. They were incubated overnight at 37 °C.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								After successful NheI digestion, BRET parts 2 and 3 were ligated to each other. This was done to be able to perform a 1:1 ligation instead of a 2:1 ligation.
+								However, the agarose gel made after ligation did not show any successful results.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal105">
+                    Tuesday August 20, 2019 - Small culture & digestion
+            </div>
+                <div class="modal" id="modal105">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday August 20, 2019 - Small culture & digestion</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+                            <p>
+								Transformation was successful, so small cultures were made using kanamycin and LB medium. From each plate 1 colony was taken, so 2 small cultures containing Sbit
+								and 2 small cultures containing Lbit. In addition, a positive control was made containing just a pipette tip without tipping a colony.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Since ligation of part 2 and 3 was not successful, digestion was performed again to prepare the parts for 2:1 ligation (part 2 and 3 separately into the
+								digested vector). Part 2 was restricted with SacI & NheI restriction enzymes and part 3 was restricted with NheI & XhoI restriction enzymes. In addition,
+								the vector was digested using the SacI & XhoI restriction enzymes. The digested samples were then ran on an agarose gel and cut out.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal106">
+                    Wednesay August 21, 2019 - Large culture, ligation & digestion
+            </div>
+                <div class="modal" id="modal106">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesay August 21, 2019 - Large culture, ligation & digestion</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+                            <p>
+L large cultures were made from 1 Sbit and 1 Lbit small culture. The bacteria were grown at 37 °C and 160 rpm until OD600 = 0.6-0.7. At OD600 = 0.6, protein
+								expression was initiated by adding IPTG. The cultures were then grown overnight at 18 °C and 160 rpm.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Gel extraction was performed for the digested part 2 and 3 and the digested vector. A 2:1 ligation was performed to ligate digested parts 2 and 3 into the digested
+								vector. The ligation product was transformed into NovaBlue cells.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal107">
+                    Thursday August 22, 2019 - Purification, PCR
+            </div>
+                <div class="modal" id="modal107">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday August 22, 2019 - Purification, PCR</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+                            <p>
+								The large cultures were centrifuged and the pellets containing bacteria were lysed using ultrasound. After this the samples were centrifuged again to spin
+								down the lysed bacteria. The lysate was purified with a strep column. The elution was captured in 1.5 ml fractions and protein concentrations were measured
+								with NanoDrop.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Transformation was successful. 14 colonies were selected for colony PCR and this was put on an agarose gel. Colonies 1, 2, 3, 4, 7, 8, 9, 10,
+, 12 & 14 seem to be successful. 14 small cultures were made with all selected colonies. In addition, parts 1 and 2 were PCR amplified using
+								overhang PCR and the designed primers to prepare them for Gibson Assembly.
+							</p>
+							<h3>Peking Split Luciferase</h3>
+							<p>
+								The inserts which were ordered at Twist Biosience were multiplied with PCR, because we will need 1 μg for digestion. These inserts will be ligated
+								in our own vector after dCas9. The sequence for our linker, NanoLuc small bit and NanoLuc large bit will be cut out.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal108">
+                    Friday August 23, 2019 - SDS & Bioluminescence test
+            </div>
+                <div class="modal" id="modal108">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday August 23, 2019 - SDS & Bioluminescence test </h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								To determine the purity of the samples that were purified a SDS page gel was made. Furthermore, Lbit of NanoLuc is slightly bioluminescent
+								itself. This was tested with the plate reader.
+                            </p>
+							<h3>BRET</h3>
+							<p>
+								The small cultures were miniprepped and the DNA concentrations were measured using NanoDrop.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal109">
+                    Monday August 26, 2019 - Transformation
+            </div>
+                <div class="modal" id="modal109">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday August 26, 2019 - Transformation</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We ordered three gBlocks containing target DNA at IDT for testing. We PCR amplified these gBlocks to use them for all bioluminescence tests.
+								After PCR amplification and PCR purification, we measured the DNA concentrations using NanoDrop.
+                            </p>
+							<h3>BRET</h3>
+							<p>
+								The successfully ligated DNA for our BRET sensor was transformed into BL21 cells and plated out. The plates were incubated overnight at 37 °C.
+								In addition, LB medium for large cultures was prepared for expression later this week.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal110">
+                    Tuesday August 27, 2019 - Sequencing & purification
+            </div>
+                <div class="modal" id="modal110">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday August 27, 2019 - Sequencing & purification</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								The second batch of dCa9s-Lbit and dCas9-Sbit were purified with strap tag columns. The concentrations were measured using NanoDrop.
+								Transformation into NovaBlue was done, because there was not enough DNA for sequencing samples.
+                            </p>
+							<h3>BRET</h3>
+							<p>
+								Sequencing samples were made to confirm the ligation was successful. The samples were sent to BaseClear for sequencing. Furthermore,
+								colonies were picked and small cultures were made.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal111">
+                    Wednesday August 28, 2019 - Small cultures & SDS gel
+            </div>
+                <div class="modal" id="modal111">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday August 28, 2019 - Small cultures & SDS gel</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								A SDS gel was made to confirm the purification of yesterday. Furthermore, NB small cultures were made which will be miniprepped tomorrow.
+								As you can see, a big blob can be seen for the elution fractions. A band would be expected at 179 kDa for Lbit and at 163 kDa for Sbit.
+                            </p>
+							<h3>BRET</h3>
+							<p>
+								The large culture media turned out to be infected. New culture media and new small cultures were made.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal112">
+                    Thursday August 29, 2019 - Expression & Q-Tof sample
+            </div>
+                <div class="modal" id="modal112">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday August 29, 2019 - Expression & Q-Tof sample</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								A Q-Tof sample was made for Split NL. However, our proteins are too big for Q-Tof, so no results were retrieved.
+								In addition, miniprepping of the small cultures was done to retrieve more DNA for sequencing. The DNA concentrations were measured using NanoDrop.
+                            </p>
+							<h3>BRET</h3>
+							<p>
+								The small cultures were added to the large cultures (1.5L) after adding kanamycin. Expression was initiated at an OD of approximately 0.6 with 200 μL/L IPTG.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal113">
+                    Friday August 30, 2019 - Purification
+            </div>
+                <div class="modal" id="modal113">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday August 30, 2019 - Purification</h2></div>
+                        <div class="modal-body">
+							<h3>BRET</h3>
+							<p>
+								The large cultures were spinned down in 2 bottles per culture and lysed using sonication. Per large culture 1 bottle was purified and the other lysed sample was
+								stored at -80 °C. His and Strep purification was performed. After His purification, no protein was present in the eluted samples.
+							</p>
+                        </div>
+                    </div>
+                </div>
+            </br>
+			<h3>September</h3>
+			<div class="box middle notebook" data-modal="modal114">
+                    Monday September 2, 2019 - Testing
+            </div>
+                <div class="modal" id="modal114">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday September 2, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								Started the first tests with the dCas9-Split-NanoLuc sensor. We tested with 50 μL samples in a 384-well plate:
+								<ol class="list">
+									<li>Only PBS + 0.1% BSA</li>
+									<li>10 nM SmallBit</li>
+									<li>10 nM LargeBit</li>
+									<li>10 nM SmallBit + 10 nM LargeBit</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + 1.2nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM unspecific DNA</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 1.2 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 1.2 nM DNA 2</li>
+								</ol>
+							</p>
+							<p>
+								NanoGlo in 1000x dilution was added to each sample before testing.
+							</p>
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Last Friday, no protein was present after His purification. Since we circularly permutated the dCas9 protein, it would be possible that the His tag
+								is not available. Therefore, we decided to only perform Strep purification using the His flow-through from last time. We measured the concentrations
+								using NanoDrop.
+							</p>
+							<h3>Peking</h3>
+							<p>
+								To be able to compare our new sensor with the sensor from Peking 2015, we decided to produce the Peking sensor containing Firefly luciferase instead
+								of Split NanoLuc. We ordered the sequences of the split firefly luciferases with their linker and will put these insert behind our own dCas9. First
+								digestion was performed with the original vector, Cluc insert, Nluc insert. PCR purification was done and the concentrations were measured using NanoDrop.
+								The samples were put on an agarose gel and the correct bands were cut out.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal115">
+                    Tuesday September 3, 2019 - Testing
+            </div>
+                <div class="modal" id="modal115">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday September 3, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We did more testing in triplo with 50 μL samples in a 384-well plate:
+								<ol class="list">
+									<li>Only PBS + 0.1% BSA</li>
+									<li>10 nM SmallBit</li>
+									<li>10 nM LargeBit</li>
+									<li>10 nM SmallBit + 10 nM LargeBit</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + 1.2nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM unspecific DNA</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 0.6 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 2.4 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 0.6 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 1.2 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 2.4 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 0.6 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 1.2 nM DNA 2</li>
+									<li>10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 2.4 nM DNA 2</li>
+								</ol>
+							</p>
+							<p>
+								NanoGlo in 2000x dilution was added to each sample before testing.
+							</p>
+							</p>
+							<p>
+								In the afternoon, we tested with lower sensor concentrations in duplo:
+								<ol class="list">
+									<li>Only PBS + 0.1% BSA</li>
+									<li>1 nM SmallBit + 1 nM LargeBit</li>
+									<li>0.5 nM SmallBit + 0.5 nM LargeBit</li>
+									<li>1 nM SmallBit + 1 nM LargeBit + 1.2 nM DNA 2</li>
+									<li>0.5 nM SmallBit + 0.5 nM LargeBit + 1.2 nM DNA 2</li>
+									<li>1 nM SmallBit + 1 nM LargeBit + sgRNA 2 + 1.2 nM unspecific DNA</li>
+									<li>0.5 nM SmallBit + 0.5 nM LargeBit + sgRNA 2 + 1.2 nM unspecific DNA</li>
+									<li>1 nM SmallBit + 1 nM LargeBit + sgRNA 2 + 1.2nM DNA 2</li>
+									<li>0.5 nM SmallBit + 0.5 nM LargeBit + sgRNA 2 + 1.2nM DNA 2</li>
+								</ol>
+							</p>
+							<p>
+								NanoGlo in 2000x dilution was added to each sample before testing.
+							</p>
+							When analyzing the results, the control samples 4 and 5 in test 1 and control samples 3, 4 and 5 in test 2 gave a very high signal. The signal was much higher
+							than the samples containing sgRNA and target DNA. Therefore, we put samples on the SDS gel. However, we used a relatively high concentration of BSA in the buffer,
+							which can clearly be seen on gel due to a band at the height of 50 kDa. Some smears can be observed around 300 kDa, indicating aggregation, but it cannot be seen
+							clearly due to the presence of BSA.
+							<h3>Peking</h3>
+							<p>
+								The DNA from the piece of gel which was cut out yesterday was extracted. Nluc and Cluc were each separately ligated into the vector containing dCas9. After ligation,
+								the DNA was transformed into NovaBlue.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal116">
+                    Wednesay September 4, 2019 - Colony PCR
+            </div>
+                <div class="modal" id="modal116">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesay September 4, 2019 - Colony PCR  </h2></div>
+                        <div class="modal-body">
+							<h3>Peking</h3>
+							<p>
+								Ligation was successful since colonies were present. Colony PCR was performed, but the results did not show anything. The positive result had failed as well, so we
+								cannot use it. Random colonies were selected and small cultures were made.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal117">
+                    Tuesday September 5, 2019 - Testing
+            </div>
+                <div class="modal" id="modal117">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday September 5, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We read that dCas9 shows aggregation when incubated at 37 °C, and that this can be prevented by incubation at room temperature for at least 10 minutes.
+								Therefore, we tested the sensor activity under different incubation conditions. Tests in duplo:
+								<ol class="list">
+									<li>10 nM Sbit + 10 nM Lbit | <i>incubated for 10 minutes at room temperature</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | <i>incubated for 10 minutes at room temperature</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | <i>incubated for 10 minutes at room temperature</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | <i>incubated for 10 minutes at room temperature</i></li>
+									<li>10 nM Sbit + 10 nM Lbit | <i>incubated for 30 minutes at room temperature</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | <i>incubated for 30 minutes at room temperature</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | <i>incubated for 30 minutes at room temperature</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | <i>incubated for 30 minutes at room temperature</i></li>
+									<li>10 nM Sbit + 10 nM Lbit | <i>incubated for 30 minutes at 37 °C</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | <i>incubated for 30 minutes at 37 °C</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | <i>incubated for 30 minutes at 37 °C</i></li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | <i>incubated for 30 minutes at 37 °C</i></li>
+								</ol>
+							</p>
+							<p>
+								NanoGlo in 2000x dilution was added to each sample.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								The sequencing for the BRET sensor showed a completely wrong sequence for all samples. However, when we further analyzed the sequence it completely
+								aligns with the sequence for our Split-NL sensor. When our original vector was digested using the correct restriction enzymes, the bands on the gel
+								were difficult to distinguish from each other. Probably both our original insert (of the Split-NL sensor) and the vector were present in the sliced gel.
+								Therefore, the original insert was ligated into the vector. We tested both sample 10 and 11 for luminescence and BRET sample 10 showed a big peak,
+								indicating the presence of LargeBit. BRET sample 11 did not show any luminescence.
+							</p>
+							<h3>Peking</h3>
+							<p>
+								Small cultures were miniprepped and transformed into BL21(DE3).
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal118">
+                    Monday September 9, 2019 - Small culture & Gibson
+            </div>
+                <div class="modal" id="modal118">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday September 9, 2019 - Small culture & Gibson </h2></div>
+                        <div class="modal-body">
+							<h3>BRET</h3>
+							<p>
+								Since BRET parts 10 and 11 did not show the correct sequence for sequencing, we decided to check the sequence for other colonies. We decided to check the
+								sequence for colonies 3 and 8. Small cultures in NovaBlue were made for these colonies.
+							</p>
+							<p>
+								Furthermore Gibson Assembly was performed to ligate the three G-blocks. The mixture was transformed into XL10-Gold cells and plated. The samples of the
+								Gibson reaction were put on an agarose gel.
+							</p>
+							<h3>Peking</h3>
+							<p>
+								Small cultures were made. Furthermore, large culture medium, LB medium and LB agar medium were prepared and autoclaved.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal119">
+                    Tuesday September 10, 2019 - Sequencing samples & testing
+            </div>
+                <div class="modal" id="modal119">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday September 10, 2019 - Sequencing samples & testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<h4>Sequencing</h4>
+							<p>
+								Sequencing samples were made and sent to BaseClear:
+								<ul class="list">
+									<li>Sample 1: Sbit 6 + dCas9-G247-FOR</li>
+									<li>Sample 2: Sbit 6 + dCas9-G485-FOR</li>
+									<li>Sample 3: Sbit 6 + dCas9-G717-FOR</li>
+									<li>Sample 4: Sbit 6 + dCas9-A932-FOR</li>
+									<li>Sample 5: Sbit 6 + dCas9-T1167-FOR</li>
+									<li>Sample 6: Lbit 13 + dCas9-G247-FOR</li>
+									<li>Sample 7: Lbit 13 + dCas9-G485-FOR</li>
+									<li>Sample 8: Lbit 13 + dCas9-G717-FOR</li>
+									<li>Sample 9: Lbit 13 + dCas9-A932-FOR</li>
+									<li>Sample 10: Lbit 13 + dCas9-T1167-FOR</li>
+								</ul>
+							</p>
+							<h4>testing</h4>
+							<p>
+								A test was performed where the interspace distance was varied. Incubation was done for 30 minutes at room temperature. Order of addition
+								in the plate: (1) DNA (2) Sbit (3) Lbit. NanoGlo was added in a 2000x dilution. Samples tested:
+								<ol class="list">
+									<li>10 nM Sbit + 10 nM Lbit </li>
+									<li>10 nM Sbit + 10 nM Lbit + 1.2 nM DNA 2 </li>
+									<li>10 nM Sbit + 10 nM Lbit + 1.2 nM non-target DNA </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nM DNA 1 (interspace distance 12) </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 1 (interspace distance 15) </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nMDNA 2 (interspace distance 17) </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nMDNA 2 (interspace distance 20) </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 2 (interspace distance 22) </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nM DNA 3 (interspace distance 27) </li>
+									<li>10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 3 (interspace distance 30) </li>
+								</ol>
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Miniprepping of BRET no.3 & BRET no.8 (small cultures) was done. These are 2 colonies with the correct vector length during colony PCR.
+								Afterwards, sequencing samples were made of these BRET samples and sent to BaseClear:
+								<ul class="list">
+									<li>Sample 11: BRET 10 + T7.FOR </li>
+									<li>Sample 12: BRET 10 + dCas9-S1338-FOR</li>
+									<li>Sample 13: BRET 10 + dCas9-G247-FOR</li>
+									<li>Sample 14: BRET 10 + dCas9-G485-FOR</li>
+									<li>Sample 15: BRET 10 + dCas9-G717-FOR</li>
+									<li>Sample 16: BRET 10 + dCas9-A932-FOR </li>
+									<li>Sample 17: BRET 3 + T7.FOR</li>
+									<li>Sample 18: BRET 8 + T7.FOR</li>
+								</ul>
+							</p>
+							<p>
+								No colonies were present after transformation, so Gibson Assembly failed. When looking at the gel, this is not suprising.
+							</p>
+							<h3>Peking</h3>
+							<p>
+								The small cultures were added to the large cultures. At the end of the day the cultures were induced with IPTG to activate protein expression.
+								Furthermore, new LB agar plates were made.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal120">
+                    Wednesay September 11, 2019 - Overhang PCR
+            </div>
+                <div class="modal" id="modal120">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesay September 11, 2019 - Overhang PCR</h2></div>
+                        <div class="modal-body">
+							<h3>BRET</h3>
+							<p>
+								The overhang PCR of G-block 3 had failed before. This was done again (G3Bv2) because the overhangs are needed to perform Gibson Assembly of
+								G-block 2 and 3 into the backbone (SacI-AgeI). After PCR amplification and purification, Gibson Assembly was performed. The samples for Gibson
+								were also put on an agarose gel to check whether PCR was successful.
+							</p>
+							<p>
+								G-block 2 and 3 were also digested for regular ligation. However, this had failed due to too low concentrations.
+							</p>
+							<h3>Peking</h3>
+							<p>
+								Due to many changes in our original vector we discovered that there were no tags in the Peking construct for purification. The His tag was
+								lost when the original dCas9 was replaced by the dCas9 from Addgene, due to the restriction sites. Therefore the large cultures were thrown
+								away and we decided not to use these samples anymore.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal121">
+                    Tuesday September 12, 2019 - Testing & Transformation
+            </div>
+                <div class="modal" id="modal121">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday September 12, 2019 - Testing & Transformation</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								A test was performed where the samples were incubated for 30 minutes at room temperature. Afterwards, the samples were centrifuged at 13,400 rpm for 5 minutes
+								to get rid of aggregates. The supernatant was used for testing. NanoGlo was added in 2000x dilution and samples of 30 μL were tested. Samples:
+								<ol class="list">
+									<li>10 nM Sbit + 10 nM Lbit</li>
+									<li>10 nM Sbit + 10 nM Lbit + 1.2 nM DNA 1</li>
+									<li>10 nM Sbit + 10 nM Lbit + gRNA 3 + 1.2 nM non-target DNA </li>
+									<li>10 nM Sbit + 10 nM Lbit + gRNA 1 + 1.2 nM DNA 1 (interspace distance 10) </li>
+									<li>10 nM Sbit + 10 nM Lbit + gRNA 2 + 1.2 nM DNA 1 (interspace distance 12) </li>
+									<li>10 nM Sbit + 10 nM Lbit + gRNA 3 + 1.2 nM DNA 1 (interspace distance 15)</li>
+								</ol>
+							</p>
+							<p>
+								In addition, a test was performed where the gRNA was added in a 6x excess (final concentration of 13 nM). This was based on 2 papers. The samples were incubated
+								for 30 minutes at room temperature. NanoGlo was added in 2000x dilution and samples of 30 μL were tested. Samples:
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit </li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM DNA 1 </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM non-target DNA </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 1 (interspace distance 10) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 1 (interspace distance 12) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 1 (interspace distance 15) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 2 (interspace distance 17) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 2 (interspace distance 20) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 2 (interspace distance 22) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 3 (interspace distance 25) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 3 (interspace distance 27) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 3 (interspace distance 30)</li>
+								</ol>
+							</p>
+							<h3>BRET</h3>
+							<p>
+								The Gibson Assembly mixture was transformed into XL10-Gold competent cells.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal122">
+                    Monday September 16, 2019 - Ligation & transformation
+            </div>
+                <div class="modal" id="modal122">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday September 16, 2019 - Ligation & transformation</h2></div>
+                        <div class="modal-body">
+							<h3>BRET</h3>
+							<p>
+								Transformation of the Gibson Assembly mixture had failed, since no colonies were present. We decided to do a 1 insert ligation with part 2 into the backbone.
+								In addition, a 2 insert ligation with both parts 2 and 3 was also performed.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal123">
+                   Tuesday September 17, 2019 - Ligation & transformation
+            </div>
+                <div class="modal" id="modal123">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday September 17, 2019 - Ligation & transformation</h2></div>
+                        <div class="modal-body">
+							<h3>BRET</h3>
+							<p>
+								Yesterday's 1 insert and 2 insert ligations of gBlocks BRETp2 and BRETp2 + BRETp3 respectively failed. The agar plates were devoid of colonies,
+								whereas the positive control plate was not. Therefore, the ligations were repeated with alternative molar ratios (4:1 and 5:5:1 respectively,
+								insert:(insert:)vector), starting from the same restriction digested building blocks.
+							</p>
+							<p>
+								Moreover, restriction digestions were performed to form a new set of digested gBlocks, which were subsequently purified by agarose gel.
+							</p>
+							<h3>Characterization</h3>
+							<p>
+								For a Bronze medal we need to make a contribution in the form of a characterization to an existing part in the Registry. iGEM Eindhoven
+created a CT52-mNeonGreen part they did not express due to lack of time. They expressed CT52-Sbit and CT52-Lbit, so therefore it
+								would be plausible that expression of this vector would also be possible. We found the correct DNA in a pET28a vector and transformed
+								it in BL21 cells. Two different mutants of CT52 were found, so both were transformed.
+							</p>
+							<p>
+								In addition, a NanoLuc part was registered in the Part Registry. We want to also characterize NanoLuc, so we need to express this protein
+								as well. We were able to get the DNA sequence of NanoLuc in a pET-28a vector.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal124">
+                   Wednesday September 18, 2019 - Small cultures, digestion, ligation & transformation
+            </div>
+                <div class="modal" id="modal124">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday September 18, 2019 - Small cultures, digestion, ligation & transformation   </h2></div>
+                        <div class="modal-body">
+							<h3>BRET</h3>
+							<p>
+								There were no colonies on the plates inoculated with the new ligation mixtures made yesterday, except for only a single one resulting from the BRETp2 + BRETp3 + vector
+								ligation. We suspect the ligation failed to yield the desired plasmid and will perform colony PCR tomorrow.
+							</p>
+							<p>
+								DNA was extracted from yesterday's gel slices containing the relevant digested parts (<mark class="pink">/SacI/</mark>-BRETp2-<mark class="green">/NheI/</mark>; <mark class="green">/NheI/</mark>-BRETp3-<mark class="yellow">/XhoI/</mark>;
+								/AgeI/-pET28a(+)-dCas9-Lbit-<mark class="pink">/SacI/</mark>) using the Qiagen gel extraction kit. With these, new ligation reactions were performed for ligating
+								<mark class="pink">/SacI/</mark>-BRETp2-<mark class="green">/NheI/</mark> in <mark class="green">/NheI/</mark>-pET28a(+)-dCas9-Lbit-<mark class="pink">/SacI/</mark> as well as <mark class="green">/NheI/</mark>-BRETp3-<mark class="yellow">/XhoI/</mark> in <mark class="yellow">/XhoI/</mark>-pET28a(+)-dCas9-Lbit-<mark class="green">/NheI/</mark> prepared previously.
+							</p>
+							<h3>Characterization</h3>
+							<h4>CT52-mNeonGreen</h4>
+							<p>
+								Transformation was successful for both mutant forms of CT52. Small cultures were made using kanamycin and LB medium.
+							</p>
+							<h4>NanoLuc</h4>
+							<p>
+								The vector for NanoLuc did not contain enough DNA, so we first need to replicate the plasmid DNA. Therefore, transformation in NovaBlue was performed.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal125">
+                   Thursday September 19, 2019 - Testing & expression
+            </div>
+                <div class="modal" id="modal125">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday September 19, 2019 - Testing & expression</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								It would be possible that the crRNA + tracrRNA is not working. Therefore, we decided to order complete sgRNA with IDT. Today we tested with
+								the new sgRNA. The samples were incubated for 30 minutes at 37 °C and sgRNA was added in a 6x molar excess. NanoGlo was added in a 2000x dilution.
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit | <i>5mM DTT present in PBS </i></li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM target DNA |<i> 5 mM DTT present in PBS</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + non-target DNA |5 mM DTT present in PBS </i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 10 |<i>5 mM DTT present in PBS </i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 17 | <i>5 mM DTT present in PBS</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 25 | <i>5 mM DTT present in PBS </i></li>
+									<li>2 nM Sbit + 2 nM Lbit |<i> no DTT </i></li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM target DNA | <i>no DTT</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + non-target DNA |<i> no DTT</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 10 | <i>no DTT</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 17 |<i> no DTT</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 25 |<i> no DTT</i></li>
+								</ol>
+							</p>
+							<p>
+								<bluetext><b>Native-PAGE</b></bluetext> to determine if dCas9 binds to the target sequence.
+							</p>
+							<p>
+								Native PAGE sample and running buffer were prepared. Samples were made, many controls were included (see below). Samples were loaded on the gel
+								and afterwards the gel was stained with SYBRGold to stain the DNA. Furthermore, the gel was stained with Coomassie overnight.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								No colonies were present, indicating that ligation had failed once again. The ligation of part 2 was repeated and the ligation mixtures of yesterday
+								were transformed again in NovaBlue cells. In addition, colony PCR of the single colony on the plate of 17/09 was performed. The positive control
+								showed the correct band on the gel, but the colony did not.
+							</p>
+							<h3>Characterization</h3>
+							<h4>CT52-mNeonGreen</h4>
+							<p>
+								Two 1L large cultures were made (1 for each small culture) using kanamycin and LB medium. Small cultures were added to the large cultures and
+								incubated at 37 °C and 160 rpm until OD600 = 0.6-0.7. Expression was induced at OD600 = 0.6 by adding IPTG.
+							</p>
+							<h4>NanoLuc</h4>
+							<p>
+								Transformation was not successful. The transformation of BRET ligation was not successful as well, so it would be possible that something
+								went wrong during the transformation experiment. Therefore, transformation was performed again, in BL21.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal126">
+                   Friday September 20, 2019 - Purification
+            </div>
+                <div class="modal" id="modal126">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday September 20, 2019 - Purification</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								The native PAGE gel was destained with water and images were taken using ImageQuant. However, the gel took very long to run and we encountered
+								some problems. Therefore, the gel did not run long enough to be able to distinguish between bands.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								No colonies were present after transformation of the 1:1 ligation. There were some small colonies on the 2:1 ligation plate, which will be analyzed
+								using colony PCR next week.
+							</p>
+							<h3>Characterization</h3>
+							<h4>NanoLuc</h4>
+							<p>
+								The transformation was successful.
+							</p>
+							<h4>mNeonGreen</h4>
+							<p>
+								The large cultures were centrifuged, the pellets were dissolved and lysed with BugBuster. The lysate was purified with IMAC chromatography. The
+								protein concentration were measured with NanoDrop. No protein was measured and the pellet after expression was not green, so the expression had
+								probably failed.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal127">
+                   Monday September 23, 2019 - Colony PCR & testing
+            </div>
+                <div class="modal" id="modal127">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday September 23, 2019 - Colony PCR & testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								Sequencing was not successful last week, so new sequencing samples were made.
+							</p>
+							<p>
+								In addition, additional tests were performed. A new buffer was used containing 20 mM Tris-HCl, 100 mM KCl, 5 mM MgCl<sub>2</sub>and 5% glycerol.
+μL samples were made and 2000x diluted NanoGlo was added. We used 2 different protein batches; one batch that was purified on 22-08
+								and one batch that was purified on 27-08.
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit | <i>Proteins from 22-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM DNA 1 |<i> Proteins from 22-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 |<i> Proteins from 22-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | <i>Proteins from 22-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 1 |<i> Proteins from 22-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 2|<i> Proteins from 22-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 3 |<i> Proteins from 22-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit |<i> Proteins from 27-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + DNA 1|<i> Proteins from 27-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 | <i>Proteins from 27-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA| <i>Proteins from 27-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 1 |<i> Proteins from 27-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 2 |<i> Proteins from 27-08</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 3 |<i> Proteins from 27-08</i></li>
+								</ol>
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Colony PCR was performed using the colonies from the transformation of 20/09. However, colony PCR failed
+								since the positive control showed a band at the correct height, but all samples did not show any band.
+							</p>
+							<h3>Characterization</h3>
+							<h4>mNeonGreen</h4>
+							<p>
+								Protein expression was not successful, so sequencing samples were made to check whether the DNA sequence is correct. Sequencing samples
+								were made using the T7 FOR primer. The sequencing samples showed that a part of the mNeonGreen sequence was not correct. Therefore, we
+								decided to use a different part for characterization.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal128">
+                   Tuesday September 24, 2019 - Small cultures
+            </div>
+                <div class="modal" id="modal128">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday September 24, 2019 - Small cultures</h2></div>
+                        <div class="modal-body">
+							<h3>Characterization</h3>
+							<p>
+								Small cultures were made for NanoLuc.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal129">
+                   Wednesday September 25, 2019 - Testing
+            </div>
+                <div class="modal" id="modal129">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday September 25, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We found out that we miscalculated the DNA concentration, so we have been using 6 pM DNA instead of 6 nM DNA. Therefore, we have only been measuring
+								background signal. New tests were performed using the correct DNA amounts. 30 μL samples were created with a 2 nM protein concentration, a 6x molar
+								excess of sgRNA and a 6 nM DNA concentration. The protein with sgRNA was incubated at 37 °C for 10 minutes and incubation of the complex with DNA was
+								done for 30 minutes at room temperature. For half of the samples the sgRNA was incubated at 50 °C for 5 minutes before use, which could avoid secondary
+								structure formation of the sgRNA before incubation with dCas9.
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) | <i>sgRNA incubation at 37 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) | <i>sgRNA incubation at 37 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) | <i>sgRNA incubation at 37 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | <i>sgRNA incubation at 37 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 | <i>sgRNA incubation at 37 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA </li>
+									<li>2 nM Sbit + 2 nM Lbit </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) |<i> sgRNA incubation at 50 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) |<i> sgRNA incubation at 50 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) |<i> sgRNA incubation at 50 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA |<i> sgRNA incubation at 50 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 | <i>sgRNA incubation at 50 °C</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA </li>
+									<li>2 nM Sbit + 2 nM Lbit </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 2A + sgRNA 1B + target DNA | <i>sgRNA incubation at 50 °C</i></li>
+								</ol>
+							</p>
+							<h3>BRET</h3>
+							<p>
+								After extracting the digested parts and backbones from yesterday's agarose gel using the Qiagen gel extraction kit , we tried two more
+								single insert ligations for creating our BRET sensor construct:
+								<ul class="list">
+									<li><mark class="green">/NheI/</mark>-BRETp3 -<mark class="yellow">/XhoI/</mark> in <mark class="yellow">/XhoI/</mark>-pET28a(+) -<mark class="green">/NheI/</mark></li>
+									<li><mark class="pink">/SacI/</mark>-BRETp2 -<mark class="green">/NheI/</mark> in <mark class="green">/NheI/</mark>-pET28a(+) -<mark class="pink">/SacI/</mark></li>
+								</ul>
+							</p>
+							<p>
+								Both were performed using T4 DNA ligase, in 20 and 25 μL reactions for BRETp2 and BRETp3 respectively and 50 ng backbone was used.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal130">
+                   Thursday September 26, 2019 - Testing
+            </div>
+                <div class="modal" id="modal130">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday September 26, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								More tests were performed to find the optimal interspace distance. 30 μL samples were made in triplo. The sgRNA was incubated for 10 minutes
+								at 50 °C before use. Subsequently, sgRNA and Sbit or Lbit was incubated for 10 minutes at 37 °C. The sgRNA:protein complex was incubated with
+								DNA for 30 minutes at room temperature. NanoGlo was added in 2000x dilution just before testing.
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 3 (interspace distance 30) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 3 (interspace distance 27) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 2 (interspace distance 22) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 2 (interspace distance 20) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 1 (interspace distance 15) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 1 (interspace distance 12) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) </li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA</li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 1 </li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA</li>
+									<li>2 nM Sbit + 2 nM Lbit</li>
+								</ol>
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Unfortunately, the ligation reactions tried yesterday appear to have failed, again no colonies had grown on the plates. We ordered new
+								gBlocks coding for the BRET sensor from IDT last night that are ready for Gibson assembly in our pET28a(+) vector, linearized by PCR.
+							</p>
+							<h3>Characterization</h3>
+							<p>
+								We had a meeting with one of our supervisors and we decided to characterize mCherry.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal131">
+                   Monday September 30, 2019 - Testing
+            </div>
+                <div class="modal" id="modal131">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday September 30, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								Bioluminescent tests were performed with centrifugation before testing to hopefully prevent aggregation. Samples of 30 μL
+								were made using 2 nM protein, 1.2 nM DNA and a 6x molar excess of gRNA. NanoGlo was added in 2000x dilution to each sample.
+								gRNA was incubated at 50 °C for 5 minutes prior to use. Subsequently, gRNA and protein were incubated at 37 °C for 20 minutes.
+								DNA with the protein-gRNA complex was incubated at room temperature for 45 minutes.
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit | <i>5 min centrifugation after incubation </i></li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 non-target DNA | <i>5 min centrifugation after incubation</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3| <i>5 min centrifugation after incubation</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM non-target DNA | <i>5 min centrifugation after incubation</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3 + DNA 3 | <i>5 min centrifugation after incubation</i></li>
+									<li>2 nM Sbit + 2 nM Lbit </i></li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 non-target DNA</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM non-target DNA</i></li>
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA 3 + DNA 3</i></li>
+								</ol>
+							</p>
+							<h3>Characterization preparation</h3>
+							<h4>Cysteine free NanoLuc</h4>
+							<p>
+								Transformation in BL21 was done.
+							</p>
+                        </div>
+                    </div>
+                </div>
+            </br>
+			<h3>October</h3>
+			<div class="box middle notebook" data-modal="modal132">
+                   Tuesday October 1, 2019 - Small cultures
+            </div>
+                <div class="modal" id="modal132">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday October 1, 2019 - Small cultures</h2></div>
+                        <div class="modal-body">
+							<h3>Characterization preparation</h3>
+							<h4>Cysteine free NanoLuc</h4>
+							<p>
+								Colonies were present, so small cultures were made.
+							</p>
+							<h4>mCherry</h4>
+							<p>
+								Small cultures were made from a glycerol stock with mCherry in BL21.
+								Furthermore, large culture media were prepared for expression of cysteine free NanoLuc and mCherry.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal133">
+                   Wednesday October 2, 2019 - Expression & testing
+            </div>
+                <div class="modal" id="modal133">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday October 2, 2019 - Expression & testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We performed test with a variable protein concentrations. We used the concentrations 2 nM, 1 nM and 0.5 nM. We used 30 μL samples using the Tris-HCl
+								buffer containing BSA and DTT. DNA concentrations of 1.2 nM and a gDNA 6x molar excess were used. gRNA was incubated at 50 °C for 10 minutes.
+								Incubation of Sbit and Lbit with gRNA was performed for 20 minutes at 37 °C, and subsequently incubation with DNA was done for 45 minutes at room
+								temperature. NanoGlo was added in 2000x excess.
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit</li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA</li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA</li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA + 1.2 nM nontarget DNA </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA + 1.2 nM DNA 3 (interspace distance 30) </li>
+									<li>1 nM Sbit + 1 nM Lbit</li>
+									<li>1 nM Sbit + 1 nM Lbit + 1.2 nM nontarget DNA</li>
+									<li>1 nM Sbit + 1 nM Lbit + gRNA</li>
+									<li>1 nM Sbit + 1 nM Lbit + gRNA + 1.2 nM nontarget DNA </li>
+									<li>1 nM Sbit + 1 nM Lbit + gRNA + 1.2 nm DNA 3 (interspace distance 30) </li>
+									<li>0.5 nM Sbit + 0.5 nM Lbit </li>
+									<li>0.5 nM Sbit + 0.5 nM Lbit + 1.2 nM nontarget DNA</li>
+									<li>0.5 nM Sbit + 0.5 nM Lbit + gRNA</li>
+									<li>0.5 nM Sbit + 0.5 nM Lbit + gRNA + 1.2 nM nontarget DNA </li>
+									<li>0.5 nM Sbit + 0.5 nM Lbit + gRNA + 1.2 nM DNA 3 (interspace distance 30) </li>
+								</ol>
+							</p>
+							<h3>Characterization</h3>
+							<h4>mCherry & Cysteine-free NanoLuc</h4>
+							<p>
+								The expression of cysteine free NanoLuc and mCherry was activated by adding 1 mM IPTG at OD 0.5. The large cultures were incubated overnight
+°C at 160 rpm.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal134">
+                   Thursday October 3, 2019 - Testing & purification
+            </div>
+                <div class="modal" id="modal134">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday October 3, 2019 - Testing & purification</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We performed tests using variable concentrations of mNeonGreen-NanoLuc to determine which concentration we would be using in our experiments later on.
+μL samples were used in the Tris-HCl, MgCl<sub>2</sub>buffer containing DTT and BSA. NanoGlo was added in 2000X dilution.
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM DNA 3 (interspace distance 30) </li>
+									<li>0.1 pM mNeonGreen-NanoLuc</li>
+									<li>0.2 pM mNeonGreen-NanoLuc</li>
+									<li>1 pM mNeonGreen-NanoLuc</li>
+									<li>2 pM mNeonGreen-NanoLuc</li>
+									<li>5 pM mNeonGreen-NanoLuc</li>
+									<li>10 pM mNeonGreen-NanoLuc</li>
+									<li>20 pM mNeonGreen-NanoLuc</li>
+								</ol>
+							</p>
+							<h3>mCherry</h3>
+							<p>
+								The large culture of mCherry was purified with Nickle affinity chromatography.
+							</p>
+							<p>
+								Fluorescence was measured.
+								<ul class="list">
+									<li>96 Well plate</li>
+									<li>A7. 1nM mCherry</li>
+									<li>A8. 1pM mCherry</li>
+									<li>A9. 10 pM mCherry</li>
+									<li>A10. 100 pM mCherry</li>
+									<li>A11. 10 nM mCherry</li>
+								</ul>
+							</p>
+							<h3>Cysteine free NanoLuc</h3>
+							<p>
+								The large culture was purified with Nickle affinity chromatography. After elution
+								a concentration of 1.36 mg/ml was measured with Nanodrop. Tomorrow the elution will be further purified with the strep tag.
+							</p>
+							<h3>Phage experiments</h3>
+							<p>
+								One of us went to the MCT lab of the QAMH Brussels to discuss with Dr. Pirnay about the experiments we would like to perform
+								with phages. A 100 μL sample of T7 phages with a high titer of 10<sup>11</sup>PFU/mL was also taken back to Eindhoven to denature by heat
+								and allow for testing our split-NL sensor on whole T7 genome.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal135">
+                   Friday October 4, 2019 - PCR for Gibson & Testing
+            </div>
+                <div class="modal" id="modal135">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday October 4, 2019 - PCR for Gibson & Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								T7 phages were heat denatured at 70 °C for 2 hours. The phage DNA was then tested. A 2 nM protein concentration, ±0.1 nM phage DNA concentration and
+x excess of gRNA was used. The gRNA was incubated at 50 °C before use. The gRNA with protein was incubated at 37 °C for 10 minutes and subsequently
+								the gRNA-protein complex was incubated with DNA for 30 minutes at room temperature. NanoGlo was then added in a 1000x dilution.
+								<ol class="list">
+									<li>2 nM Sbit + 2 nM Lbit</li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA</li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA </li>
+									<li>2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA + gRNA </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 1 + 0.1 nM T7 phage DNA (interspace distance 17) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 2 + 0.1 nM T7 phage DNA (interspace distance 20) </li>
+									<li>2 nM Sbit + 2 nM Lbit + gRNA 3 + 0.1 nM T7 phage DNA (interspace distance 22)</li>
+								</ol>
+							</p>
+							<h3>BRET</h3>
+							<p>
+								The backbone required for the new Gibson assembly strategy we will try next week were linearized from the original pET28a(+)-split-NL vector by PCR
+								in two variants:
+								<ul class="list">
+									<li>One complete backbone was made (A, ca. 5.3 kb)</li>
+									<li>The same backbone was prepared as two parts with a 20 bp mutual overlap for Gibson assembly (B and C, ca. 2.7 and 2.6 bp respectively).</li>
+								</ul>
+							</p>
+							<p>
+								Afterwards, the resulting PCR mixtures were PCR purified and samples were run on an agarose gel to check for correct PCR products.
+							</p>
+							<h3>mCherry & Cysteine free NanoLuc</h3>
+							<p>
+								The samples from the purification were put on an SDS-PAGE gel.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal136">
+                   Monday October 7, 2019 - Gibson Assembly & QAMH
+            </div>
+                <div class="modal" id="modal136">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday October 7, 2019 - Gibson Assembly & QAMH</h2></div>
+                        <div class="modal-body">
+							<h3>BRET</h3>
+							<p>
+								Gibson assembly was performed, one sample with one backbone and one sample with the backbone in 2 parts. A gibson assembly control was also included.
+								The samples were transformed in XL10-Gold and plated.
+							</p>
+							<h3>mCherry</h3>
+							<p>
+								Dilutions were made of 100 nm, 1 μM and 10 μM. The excitation and emission spectrum were measured. The effect of photobleaching after 10 min, 20 min and
+min UV light exposure was also measured.
+							</p>
+							<h3>Phage experiments at QAMH Brussels</h3>
+							<p>
+								The first day of our experimental work at the laboratory for molecular and cellular technology (MCT lab) at Queen Astrid Military Hospital (QAMH) in Brussels!
+								Today after some introduction to the lab, meeting its researchers and getting to know where to find the things we needed, we prepared the M9 based minimal medium
+								we will use for culturing the <i>E. coli B</i> DSM 613 (ATCC 11303) bacteria. Some unexpected difficulties arose, as at first we added all components together
+								directly in random order, resulting in precipitation. We adapted the protocol after some more trial and error and doing some more research to finally obtain
+								the medium we needed. We also poured some petri dish LB-Agar plates for the experiments to be performed later this week and started two 100 ml liquid overnight
+								cultures (37 °C, 120 RPM) of the <i>E coli B</i> DSM 613 (from a collaboration between Dr. Pirnay's lab and the iGEM Munich 2018 team), taken from the plate Maya
+								Merabishvili prepared for us.
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal137">
+                   Tuesday October 8, 2019 - Gibson Assembly, testing & QAMH
+            </div>
+                <div class="modal" id="modal137">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday October 8, 2019 - Gibson Assembly, testing & QAMH</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We ordered new DNA gBlocks, so we would be able to test interspace distances 30-60 bp. Tests were performed using 2 nM protein, 1.2 nM DNA and a 6x
+								molar excess of gRNA. gRNA was incubated at 50 °C before use and the gRNA and protein were incubated at 37 °C for 20 minutes. Subsequently, the gRNA-protein
+								complex was incubated with DNA for 45 minutes at room temperature. NanoGlo was added in 2000x dilution.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								No colonies were present on all the plates. The control plates also had no colonies, but we discovered that the wrong plates were used because the control was
+								not kanamycin resistant. The gBlocks were PCRed and Gibson assembly was tried again and the samples were transformed in XL10-Gold ultracompetent cells. The
+								samples from Gibson assembly yesterday were put on an agarose gel.
+							</p>
+							<h3>Phage experiments at QAMH Brussels</h3>
+							<p>
+								We planned to start the bacterial growth rate determination experiment today, yet unfortunately the overnight cultures still appeared very transparent, whereas
+								one would expect them to be rather turbid because of a high cell density. The only turbidity we could see was some sediment, which we believe was some
+								precipitated calcium-or magnesium sulfate. OD<sub>600</sub> measurements confirmed the lack of an appreciable amount of bacteria. We discussed the apparent
+								problem with others at the lab and looked into the recipe we used once more and made slight adjustments (added a trace amount of FeSO<sub>4</sub>, kept MgCl
+								out and made some with tryptone added as control) and started some small new liquid cultures using these media. We also prepared and autoclaved 400 mL
+								semisolid LB Agar (0.8%) for later use.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal138">
+                   Wednesday October 9, 2019 - Gibson Assembly, testing & QAMH
+            </div>
+                <div class="modal" id="modal138">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday October 9, 2019 - Gibson Assembly, testing & QAMH</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								To improve the signal-to-noise ratio, we decided to test with higher protein concentrations. We used interspace distance 30 for testing, since this gave the
+								highest signal in previous tests. We under the same conditions as the previous tests, but we used the protein concentrations 2, 5 and 10 nM.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								No colonies were present on all the plates. The agarose gel of the gBlocks which were PCRed showed unexpected results for part 2. The weight was lower as expected.
+								The PCR reaction was tried again. However, the same results came out of the gel.
+							</p>
+							<h3>Phage experiments at QAMH Brussels</h3>
+							<p>
+								Luckily we did not throw away our seemingly failed cultures yesterday, but kept them in the shaking incubator instead, as this morning the media of one of the
+								flasks was all turbid! OD<sub>600</sub> measurement confirmed the presence of a high bacterial cell density, with an OD<sub>600</sub> of 1.525. The other culture
+								had obtained an OD<sub>600</sub> of 0.081 and appeared slightly turbid to the naked eye, hence it was put back in the incubator. At the end of the day this
+								culture had reached an OD<sub>600</sub> of 0.604. To check the linearity of the OD<sub>600</sub> vs. cell concentration we prepared a serial two-fold dilution
+								series and measured the OD<sub>600</sub> giving the below, nicely linear, results:
+							</p>
+							<p>
+								<table class="parts-table" align="center">
+								<tr>
+									<th>Dilution</th>
+									<th>OD<sub>600</sub> culture 1</th>
+									<th>OD<sub>600</sub> culture 2
+								</tr>
+								<tr>
+									<td>1x</td>
+									<td>1.475</td>
+									<td>0.601</td>
+								</tr>
+								<tr>
+									<td>2x</td>
+									<td>0.825</td>
+									<td>0.304</td>
+								</tr>
+								<tr>
+									<td>4x</td>
+									<td>0.433</td>
+									<td>0.159</td>
+								</tr>
+								<tr>
+									<td>8x</td>
+									<td>0.224</td>
+									<td>0.075</td>
+								</tr>
+								<tr>
+									<td>16x</td>
+									<td>0.114</td>
+									<td>0.040</td>
+								</tr>
+								<tr>
+									<td>32x</td>
+									<td>0.057</td>
+									<td>0.019</td>
+								</tr>
+								<tr>
+									<td>20x</td>
+									<td>0.090</td>
+									<td></td>
+								</tr>
+								</table>
+							</p>
+							<div class="photo-center">
+								<img src="https://2019.igem.org/wiki/images/e/e3/T--TU_Eindhoven--Lab20191009Linearity.png" alt="Process" width="100%" />
+								</br>Figure 3: linearity of the OD<sub>600</sub> vs. cell concentration of culture 1 with R<sup>2</sup> = 0.9934 and culture 2 with R<sup>2</sup> = 0.9997.
+							</div>
+							</br>
+							<p>
+							We then started the bacterial growth rate determination experiment according to the protocol. Eight 70 mL cultures were used, being 20x dilutions of the culture
+							described above, resulting in a starting OD<sub>600</sub> of 0.13 (measured in flask 8 just before start of the actual experiment). The 8 cultures differed in
+							starting concentration of glucose:
+							<ol class="list">
+								<li>0.0 g/L Glucose</li>
+								<li>0.01 g/L Glucose</li>
+								<li>0.25 g/L Glucose</li>
+								<li>0.5 g/L Glucose</li>
+								<li>1.0 g/L Glucose</li>
+								<li>2.0 g/L Glucose</li>
+								<li>4.0 g/L Glucose</li>
+								<li>8.0 g/L Glucose</li>
+							</ol>
+							</p>
+							<p>
+								A 1 mL sample was taken every 20 minutes to measure the OD<sub>600</sub> of each individual culture. At the same time, another 1 mL sample was stored in an
+								Eppendorf tube and quickly frozen using liquid nitrogen to be analyzed for remaining glucose concentration later on. A set of calibration samples was also
+								prepared covering the starting glucose concentrations of the experimental cultures and included <i>E. coli</i> at an OD<sub>600</sub> of 0.163.
+							</p>
+							<div class="photo-center">
+								<img src="https://2019.igem.org/wiki/images/2/25/T--TU_Eindhoven--Lab20191009BacterialGrowthVSGlucose.png" alt="Process" width="90%" />
+							</div>
+							</br>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal139">
+                   Thursday October 10, 2019 - Transformation, testing & QAMH
+            </div>
+                <div class="modal" id="modal139">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday October 10, 2019 - Transformation, testing & QAMH</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We tested multiple protein concentrations to see what range can be used for testing. We expected that the background signal would increase for high protein
+								concentrations. Yesterday we saw that 10 nM protein concentration gave a good signal-to-noise ratio. Today we tested 2, 10, 20, 50 and 100 nM.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Gibson assembly was tried again from the original gBlock stocks and the samples were transformed in XL10-Gold ultracompetent cells. Two controls were includes;
+								one for the Gibson assembly and one for the transformation.
+							</p>
+							<h3>Phage experiments at QAMH Brussels</h3>
+							<p>
+								Yesterday's glucose concentration calibration samples were analyzed using a Roche Hitachi cobas 6000 / c 501 (set at blood serum and GLUC-3 analysis setting)
+								made available to us at QAMH. From the results, it became clear that calibration wasn't needed as reported concentration values closely matched the expected
+								values. Due to a lack of time, the experimental samples will be analyzed tomorrow.
+							</p>
+							<p>
+								We also performed a first trial of the experiment for determination of the bacteriophage adsorption rate. A dilution of a mid-log phase culture
+								(OD<sub>600</sub> 0.549) was made (resulting in OD<sub>600</sub> 0.188) and used for the actual experiment. We performed the experiment with two different
+								phage titers in parallel, both with a final dilution of 10<sup>-7</sup> and 10<sup>-6</sup> of the 3∙10<sup>11</sup> stock. The resulting solutions were
+								plated and incubated overnight.
+							</p>
+							<p>
+								Yesterday's plates for enumerating the amount of colonies to calculate the bacteria titer showed inconsistent results, hence we will make multiple sets of
+								new plates in the upcoming days to be able to convert OD<sub>600</sub>to CFU/ml reliably.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal140">
+                   Friday October 11, 2019 - Testing
+            </div>
+                <div class="modal" id="modal140">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday October 11, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We tested the limit of detection of our sensor using 2 nM and 10 nM protein. A range of DNA concentrations from 1 pM to 10 nM was tested. The incubation
+								conditions were the same as in previous tests and NanoGlo was added in 2000x dilution.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Colonies were present one of the Gibson assembly samples, the control for Gibson and the control for transformation! The colonies from Gibson were very small
+								at first, so they were put back into the stove and taken out at the end of the day.
+							</p>
+							<h3>Phage experiments at QAMH Brussels</h3>
+							<p>
+								This morning we found our very first phage plaques! The plaques of yesterday's 24 plates were counted, but unfortunately no clear decline in unadsorbed phage
+								could be seen over the 10 minutes sampling time. Therefore, we decided to plate the samples we stored in the fridge again today, to see if the unexpected
+								result might be due to plating errors. Only the samples from the 10<sup>-6</sup> dilution were plated again, and using 200μL instead of 100μL this time to
+								increase the number of plaques, aiding counting. We also plated a 10-fold serial dilution series of the T7 phage stock in order to re-titer the stock. Later
+								the same day we could already count the plaques, but unfortunately we found the same overall result for the adsorption rate experiment. For the stock we found:
+							</p>
+								<table class="parts-table" align="center">
+								<tr>
+									<th>Dilution</th>
+									<th>Plaques</th>
+									<th>PFU/ml</th>
+								</tr>
+								<tr>
+									<td>10<sup>-7</sup></td>
+									<td>518</td>
+									<td>5E<sup>+10</sup></td>
+								</tr>
+								<tr>
+									<td>10<sup>-7</sup></td>
+									<td>68</td>
+									<td>7E<sup>+10</sup></td>
+								</tr>
+								<tr>
+									<td>10<sup>-7</sup></td>
+									<td>9</td>
+									<td>9E<sup>+10</sup></td>
+								</tr>
+								<tr>
+									<td>Average</td>
+									<td></td>
+									<td>7E<sup>+10</sup></td>
+								</tr>
+								</table>
+							<p>
+								The glucose concentrations of the samples of 9 October were also measured using the Hitachi Roche cobas 6000. The results showed some dubious deviations from
+								the expected curve, but we will still try to use it to fit our model.
+							</p>
+							<p>
+								We also performed the infection assay experiment, first growing a diluted overnight culture, starting from OD<sub>600</sub> 0.104, reaching 0.126 when starting
+								the actual experiment. To a 66 mL culture, 7*10<sup>6</sup>PFU/ml phage was added, resulting in a 7*10<sup>4</sup>PFU/ml solution. 3.8 ml growth medium and 15
+								droplets of chloroform were added in multiple falcon tubes to collect samples of 0.2 ml cell/phage suspension in. Moreover, 1 ml samples were collected in
+								cuvettes for OD<sub>600</sub> measurements as well as 1 ml samples for glucose concentration measurements. The glucose samples will be analyzed next week,
+								while part of the chloroformed samples were already plated (plaque assay) in various dilutions. The remainder will be plated next week.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal141">
+                   Monday October 14, 2019 - Testing, small cultures & miniprepping
+            </div>
+                <div class="modal" id="modal141">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday October 14, 2019 - Testing, small cultures & miniprepping</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								Tests using 1 nM and 0.5 nM protein concentrations were performed to test whether the limit of detection could be lowered. DNA concentrations from 0.5 pM to
+pM were used and the same incubation conditions were used as in previous tests.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								Colony PCR was performed and small cultures were made early in the morning. The small cultures were miniprepped at the end of the day. However, some samples
+								had no DNA at all.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal142">
+                   Tuesday October 15, 2019 - Testing & PCR
+            </div>
+                <div class="modal" id="modal142">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Tuesday October 15, 2019 - Testing & PCR</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We wanted to make a calibration curve using NanoLuc-mNeonGreen. We tested a range of DNA concentrations using just our sensor without NanoLuc-mNeonGreen and
+								we tested the same range of DNA concentrations with the addition of NanoLuc-mNeonGreen.
+							</p>
+							<h3>BRET</h3>
+							<p>
+								To confirm the negative colony PCR, a regular PCR reaction was performed to check the ligation of the gBlocks. T7 reverse and forward primers were also used
+								the look at what is inside the vector.
+							</p>
+							<h3>Phage experiments at QAMH Brussels</h3>
+							<p>
+								The plaques of last Friday's plates were counted and the remaining samples that were not yet plated, were plated at several relevant dilutions. Later that day
+								we could already count the plaques on the plates with a countable number of plaques. Unfortunately, not of all sampling times the titer could be determined,
+								but with the available datapoints we found a clear overall pattern that matched our expectations.
+							</p>
+								<table class="parts-table" align="center">
+								<tr>
+									<th>Time (min)</th>
+									<th>Phage [PFU/ml]</th>
+								</tr>
+								<tr>
+									<td>0</td>
+									<td>7.00E<sup>+3</sup></td>
+								</tr>
+								<tr>
+									<td>20</td>
+									<td>3.67E<sup>+03</sup></td>
+								</tr>
+								<tr>
+									<td>40</td>
+									<td>6.67E<sup>+03</sup></td>
+								</tr>
+								<tr>
+									<td>60</td>
+									<td>9.41E<sup>+04</sup></td>
+								</tr>
+								<tr>
+									<td>80</td>
+									<td>9.99E<sup>+05</sup></td>
+								</tr>
+								<tr>
+									<td>120</td>
+									<td>1.80E<sup>+07</sup></td>
+								</tr>
+								<tr>
+									<td>160</td>
+									<td>4.60E<sup>+07</sup></td>
+								</tr>
+								<tr>
+									<td>180</td>
+									<td>4.00E<sup>+07</sup></td>
+								</tr>
+								<tr>
+									<td>200</td>
+									<td>7.00E<sup>+07</sup></td>
+								</tr>
+								<tr>
+									<td>220</td>
+									<td>3.49E<sup>+08</sup></td>
+								</tr>
+								</table>
+							</br>
+							<div class="photo-center">
+								<img src="https://2019.igem.org/wiki/images/5/5e/T--TU_Eindhoven--Lab20191015PhageGrowth.jpg" alt="Process" width="60%" />
+								</br>Figure 5: Phage growth plotted over time.
+							</div>
+							</br>
+							<p>
+								Griet Steurs helped us out by starting a bacterial culture from the plated DSM 613 for us on Sunday already. This culture was subcultured and grown for use in
+								a repetition of the adsorption rate determination experiment. Unfortunately the bacteria appeared to grow only very slowly, so we didn't perform the experiment
+								and instead started some additional liquid cultures for use tomorrow.
+							</p>
+							<p>
+								We also heat denatured (70°C, 2 hours) a 10-fold serial dilution (from 10<sup>0</sup> to 10<sup>-9</sup>) of the phage stock for experiments in Eindhoven, to try
+								to detect the freed phage DNA.
+							</p>
+							<p>
+								The glucose measurement samples collected last Friday during the infection assay were also measured using the Roche Hitachi cobas 6000.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal143">
+                   Wednesday October 16, 2019 - Testing
+            </div>
+                <div class="modal" id="modal143">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Wednesday October 16, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								The proof-of-concept was tested using denatured phages that had infected bacteria for a variable amount of time. However, we did not exactly know the DNA
+								concentration of these samples. Bioluminescent tests were performed using 2 nM sensor protein and a 3x molar excess of gRNA. The results show that we are
+								able to detect the phage DNA after infection and denaturation. However, no difference in signal can be observed for the different samples. We do not yet know
+								the cause, but it would be possible that the DNA concentration does not vary much between the samples.
+							</p>
+							<h3>Phage experiments at QAMH Brussels</h3>
+							<p>
+								A successfully grown overnight culture was used to repeat the phage adsorption rate determination experiment, with a start OD<sub>600</sub> of 0.308 and phage
+								titer 7∙10<sup>4</sup>. After discussing the results of the previous trial of this experiment with Dr. Pirnay and Dr. Merabishvili, we discovered we hadn't
+								strictly controlled the temperature, which could have influenced the adsorption. Therefore, this time we used a water bath in which the flask could remain fixed.
+							</p>
+							<p>
+								We also took pictures of all our plates that we accumulated thus far, one examples clearly showing plaques is shown below.
+							</p>
+							<div class="photo-center">
+								<img src="https://2019.igem.org/wiki/images/0/09/T--TU_Eindhoven--Lab20191016PlaquePlate.jpg" alt="Process" width="50%" />
+								</br>Figure 6: One of the accumulated plates.
+							</div>
+							</br>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal144">
+                   Thursday October 17, 2019 - Testing
+            </div>
+                <div class="modal" id="modal144">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Thursday October 17, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								We performed bioluminescence tests to check whether the limit of detection can be lowered when multiple sensor protein pairs bind to multiple recognition
+								sites on one target. We used the test gBlocks and all three gRNA pairs to test the bioluminescent signal for a range of DNA concentrations when three sensor
+								proteins bind to one target. Results show that the limit of detection can be lowered to 5 pM in this situation.
+							</p>
+							<h3>Phage experiments at QAMH Brussels</h3>
+							<p>
+								Unfortunately, all plates of the adsorption rate determination experiment contained an uncountable number of phages, in fact only a web like structure
+								remained with some resistant bacterial colonies. Clearly an error was made, as more phages than expected were present.
+							</p>
+							<p>
+								Some plates of samples of the infection assay of last week that were plated yesterday did show countable numbers of plaques, consistent over multiple dilutions.
+								Results obtained from these plates were added to the ones obtained previously (see 15-10-2019).
+							</p>
+							<p>
+								The adsorption rate determination experiment was performed one last time, over a period of 20 minutes this time. Starting OD<sub>600</sub>was 0.096, with an
+								OD<sub>600</sub> 0.142 reached 30 minutes after finishing a sampling period of 20 minutes. Note: The origin culture grew remarkably well this time, obtaining
+								an OD<sub>600</sub> of 1.237 from 0.962 in 1,25 hours. A 10-fold serial dilution series was made of this culture and plated to convert the OD<sub>600</sub>
+								values to CFU/ml.
+							</p>
+							<p>
+								The next day, Griet Steurs counted all plates for us (for which many thanks!). Unfortunately, the results of the adsorption rate determination experiment were
+								inconsistent again and didn’t show the expected decrease in unadsorbed phage. The plates for determining the cell titer were nicely consistent over multiple
+								dilutions and duplo's however, so we could reliably convert all OD<sub>600</sub> values.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal145">
+                   Friday October 18, 2019 - Testing
+            </div>
+                <div class="modal" id="modal145">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Friday October 18, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								Three different bioluminescence tests were performed: variable interspace distance, a range of phage DNA concentrations and a calibration test using
+								NanoLuc-mNeonGreen.
+							</p>
+							<h4>Variable interspace distance</h4>
+							<p>
+								We performed bioluminescence tests using interspace distances from 20 - 150 bp. A 2 nM protein concentration, 3x molar excess of gRNA and 250 pM DNA
+								concentration was used. The same incubation conditions as in previous experiments were used. The results showed a high bioluminescent signal for the
+								controls and the bioluminescent signal for many samples was lower than the controls. Therefore, we decided to perform this test again on Monday.
+							</p>
+							<h4>Range of phage DNA concentrations</h4>
+							<p>
+								From Brussels we retrieved phage samples containing a range of CFU/mL. These samples were denatured by heating them for 2 hours at 70 °C and measured
+								using our sensor. The results show a decrease in bioluminescent signal when the CFU/mL is lower. However, some outliers were present.
+							</p>
+							<h4>Calibration test using NanoLuc-mNeonGreen</h4>
+							<p>
+								We already performed a calibration test, which had quite good results. However, we did not test enough concentrations to be able to make a good calibration
+								curve containing many data points. Therefore, we did a calibration test using 2 nM sensor concentration, a 3x molar excess of gRNA, 2 pM NanoLuc-mNeonGreen
+								and a range of DNA concentrations. However, the resulting data points could not be fitted into a good calibration curve.
+							</p>
+                        </div>
+                    </div>
+                </div>
+			<div class="box middle notebook" data-modal="modal146">
+                   Monday October 21, 2019 - Testing
+            </div>
+                <div class="modal" id="modal146">
+                    <div class="modal-content">
+                        <div class="modal-header"><button class="icon modal-close">&times;</button><h2>Monday October 21, 2019 - Testing</h2></div>
+                        <div class="modal-body">
+							<h3>Split-NL</h3>
+							<p>
+								Since the test using variable interspace distances was not successful last week, we performed the test again today. We used 1.2 nM DNA concentrations instead
+								of 250 pM. The same incubation conditions and concentrations were used. We retrieved good results with an optimal interspace distance of 70 bp.
+							</p>
+                        </div>
+                    </div>
+                </div>
+            </div>
+            </div>
@@ Line 430: / Line 2,339: @@
              <div class="column middle">
                  <h2>Milestones</h2>
+                <p>
+                    Throughout the competition, a lot of milestones were achieved inside and outside the lab. Below you can find the list of the most important milestones we achieved together with our team.
+                </p>
+                </br>
                  <div class="box middle">
                      February 6, 2019 - Kick-off meeting
@@ Line 526: / Line 2,438: @@
                  <div class="box middle">
                      October 2, 2019 - Arrival Postcards Duesseldorf collaboration
+                </div>
+                <div class="box middle">
+                    October 7, 2019 - Performing our phage experiments at the QAMH in Brussels
                  </div>
                  <div class="box middle">
                      October 22, 2019 - Successful Wiki Freeze!
                  </div>
                  <div class="box middle">
                      October 25, 2019 - BeNeLux Mini Jamboree, Eindhoven

Difference between revisions of "Team:TU Eindhoven/Notebook"

Latest revision as of 03:08, 22 October 2019

Notebook

Lab Notebook

June

Monday June 17, 2019 - Stocks

Tuesday June 18, 2019 - Transformation

Wednesday June 19, 2019 - Small culture

Thursday June 20, 2019 - Miniprep & protein expression

Friday June 21, 2019 - Digestion & protein pellets

Monday June 24, 2019 - Ligation & column preparation

Tuesday June 25, 2019 - Purification & small culture

Wednesday June 26, 2019 - Miniprep & SDS-PAGE

Thursday June 27, 2019 - SDS-PAGE, sequencing & new LB stock

Friday June 28, 2019 - 2nd Transformation Sbit, IPTG stock & New plan expression

New plan Expression

July

Monday July 1, 2019 - Small culture

Split-NL

Tuesday July 2, 2019 - Small culture

Split-NL

Friday June 28, 2019 - Expression

Split-NL

Thursday July 4, 2019 - Centrifugation

Split-NL

Friday July 5, 2019 - Sonication lysis & protein purification

Split-NL

Monday July 8, 2019 - SDS-PAGE

Split-NL

Tuesday July 9, 2019 - SDS-PAGE

Split-NL

Peking constructs

Wednesday July 10, 2019 - Denaturing His-tag purification

Split-NL

Thursday July 11, 2019 - Denaturation

Split-NL

BRET

Friday July 12, 2019 - Design

Monday July 15, 2019 - General

July 15 - 19, 2019

Monday July 22, 2019 - Small culture

Split-NL

Tuesday July 23, 2019 - Miniprep & transformation

Split-NL

Wednesday July 24, 2019 - Small culture

Split-NL

Thursday July 25, 2019 - PCR, digestion & expression

Split-NL

BRET

Friday July 26, 2019 - Centrifugation, lysis, SDS-page,...

Split-NL

BRET

Monday July 29, 2019 - DNA struggles

July 29 - August 2

Split-NL

BRET

August

Monday August 5, 2019 - More DNA struggles, success for Split-NL

Split-NL

BRET

Tuesday August 13, 2019 - Reorganization

Wednesday August 14, 2019 - Digestion, PCR amplification

Split-NL

BRET

Thursday August 15, 2019 - Meeting with supervisors

General

BRET

Friday August 16, 2019 - Gibson Assembly primers design

BRET

Monday August 19, 2019 - Transformation & ligation

Split-NL

BRET

Tuesday August 20, 2019 - Small culture & digestion

Split-NL

BRET

Wednesay August 21, 2019 - Large culture, ligation & digestion

Split-NL

BRET

Thursday August 22, 2019 - Purification, PCR

Split-NL

Friday June 28, 2019 - 2^nd Transformation Sbit, IPTG stock & New plan expression