The general stocks (IPTG, Kanamycin, LB medium and LB agar for the plates) were prepared.

The vector ordered at Genscript (pET28a(+)) with our insert dCas9-Sbit-Lbit was dissolved and transformed into BL21 and NovaBlue. The vector transformed into BL21 will be used for proteins expression of dCas9-Sbit. The vector transformed into NovaBlue will be used for restriction and ligation to form dCas9-Lbit. In the end the bacteria were plated and incubated overnight.

The plates were taken out of the incubator and were put in the fridge. Not that many colonies were formed, however there were enough colonies for the small cultures. In the afternoon the small cultures were prepared and incubated overnight. Furthermore, the media for the large cultures were prepared. We do not know yet which medium will be most optimal for protein expression, so LB, 2YT and TB medium were prepared.

Figure 1: Culture plates.

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: 215.8 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.

After the two small cultures on ice were at room temperature, they were divided over the three large cultures (LB, 2YT, TB). When the OD600 had reached 0.6-0.7, protein expression was initiated by adding IPTG. The large cultures were incubated overnight.

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.

Furthermore, the DNA retrieved yesterday (miniprep) was digested to form the dCas9-Lbit. The digested DNA was put on an agarose gel 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.

The digested DNA (from 21-06) was ligated, transformed into BL21(DE3) bacteria (for protein expression) and plated. The plates are incubated overnight (dCas9-Lbit). Furthermore, the strep buffer W and IMAC columns were prepared for protein purification (of dCas9-Sbit), which will be done tomorrow. The Strep columns will be used from Eva van Aalen (PhD supervisor).

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 IMAC columns. The elution from the IMAC columns were then put on the strep columns. 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. Unfortunately there were no proteins after the purification. The question is if the proteins were expressed. Samples were taken during the whole purification process, these will be put on an SDS-page gel tomorrow to check if the protein was lost during the purification. If this is not the case, the expression protocol needs to be further looked into to optimize it for this protein.

NcoI–His tag–GGS– SacI–dCas9–AgeI–(GGS)5–HindIII–SpeI–SBit–BamHI–(GGS)2–Strep tag–stop

Ten small cultures were also made for the dCas9-Lbit which was ligated and transformed in NovaBlue yesterday.

The small cultures were taken out of the incubator and the DNA for the dCas9-Lbit was isolated using the QIAprep miniprep kit. The final DNA concentrations are shown below. Furthermore, SDS-PAGE samples were made from the flow through samples from the purification proces; His bind, His wash, Strep lysate, Strep wash, Strep elution. The samples were loaded on the gel to evaluate the purification steps. To be able to evaluate the results, the gel was washed and stained with Coomassie for approximately 40 minutes. After this, the gel is washed in water overnight on the shaking table.

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

Split-NL

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.

Split-NL

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.

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:

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.

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

Split-NL

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 H₂O 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).

Split-NL

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.

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.

Split-NL

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.

Split-NL

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.

Peking constructs

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.

Split-NL

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.

Split-NL

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.

BRET

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.

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.

July 15 - 19, 2019

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.

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.

Split-NL

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).

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:

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.

Split-NL

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).

Split-NL

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

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.

Split-NL

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.

The PCRed insert was digested, as was the His-MBP gBlock to be coupled to the dCas9 in the plasmid.

BRET

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.

July 29 - August 2

Split-NL

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.

BRET

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.

Split-NL

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.

BRET

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.

We reorganized the blue boxes containing all DNA, enzymes and reagents in the -20 °C freezer.

Split-NL

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.

BRET

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.

General

Today we had a meeting with our supervisors. We presented our progress and discussed about some problems we encountered in the lab.

BRET

We performed gel extraction of BRET part 2 and BRET part 3 after NheI digestion. The DNA concentrations were measured using NanoDrop.

BRET

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.

Split-NL

After successful ligation of the Liu Addgene dCas9 sequence into our original vector, the plasmid was transformed into BL21 cells. We used four plates: 2 for Sbit and 2 for Lbit. They were incubated overnight at 37 °C.

BRET

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.

Split-NL

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.

BRET

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.

Split-NL

2 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.

BRET

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.

Split-NL

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.

BRET

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, 11, 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.

Peking Split Luciferase

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.

Split-NL

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.

BRET

The small cultures were miniprepped and the DNA concentrations were measured using NanoDrop.

Split-NL

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.

BRET

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.

Split-NL

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.

BRET

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.

Split-NL

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.

BRET

The large culture media turned out to be infected. New culture media and new small cultures were made.

Split-NL

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.

BRET

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.

BRET

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.

Split-NL

Started the first tests with the dCas9-Split-NanoLuc sensor. We tested with 50 μL samples in a 384-well plate:

1. Only PBS + 0.1% BSA
2. 10 nM SmallBit
3. 10 nM LargeBit
4. 10 nM SmallBit + 10 nM LargeBit
5. 10 nM SmallBit + 10 nM LargeBit + 1.2nM DNA 2
6. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM unspecific DNA
7. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM DNA 2
8. 10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 1.2 nM DNA 2
9. 10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 1.2 nM DNA 2

NanoGlo in 1000x dilution was added to each sample before testing.

BRET

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.

Peking

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.

Split-NL

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

1. Only PBS + 0.1% BSA
2. 10 nM SmallBit
3. 10 nM LargeBit
4. 10 nM SmallBit + 10 nM LargeBit
5. 10 nM SmallBit + 10 nM LargeBit + 1.2nM DNA 2
6. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM unspecific DNA
7. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 0.6 nM DNA 2
8. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM DNA 2
9. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 2.4 nM DNA 2
10. 10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 0.6 nM DNA 2
11. 10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 1.2 nM DNA 2
12. 10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 2.4 nM DNA 2
13. 10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 0.6 nM DNA 2
14. 10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 1.2 nM DNA 2
15. 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:

1. Only PBS + 0.1% BSA
2. 1 nM SmallBit + 1 nM LargeBit
3. 0.5 nM SmallBit + 0.5 nM LargeBit
4. 1 nM SmallBit + 1 nM LargeBit + 1.2 nM DNA 2
5. 0.5 nM SmallBit + 0.5 nM LargeBit + 1.2 nM DNA 2
6. 1 nM SmallBit + 1 nM LargeBit + sgRNA 2 + 1.2 nM unspecific DNA
7. 0.5 nM SmallBit + 0.5 nM LargeBit + sgRNA 2 + 1.2 nM unspecific DNA
8. 1 nM SmallBit + 1 nM LargeBit + sgRNA 2 + 1.2nM DNA 2
9. 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.

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.

Peking

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.

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:

1. 10 nM Sbit + 10 nM Lbit | incubated for 10 minutes at room temperature
2. 10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | incubated for 10 minutes at room temperature
3. 10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | incubated for 10 minutes at room temperature
4. 10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | incubated for 10 minutes at room temperature
5. 10 nM Sbit + 10 nM Lbit | incubated for 30 minutes at room temperature
6. 10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | incubated for 30 minutes at room temperature
7. 10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | incubated for 30 minutes at room temperature
8. 10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | incubated for 30 minutes at room temperature
9. 10 nM Sbit + 10 nM Lbit | incubated for 30 minutes at 37 °C
10. 10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | incubated for 30 minutes at 37 °C
11. 10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | incubated for 30 minutes at 37 °C
12. 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).

BRET

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.

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.

Peking

Small cultures were made. Furthermore, large culture medium, LB medium and LB agar medium were prepared and autoclaved.

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:

1. 10 nM Sbit + 10 nM Lbit
2. 10 nM Sbit + 10 nM Lbit + 1.2 nM DNA 2
3. 10 nM Sbit + 10 nM Lbit + 1.2 nM non-target DNA
4. 10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
5. 10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
6. 10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 1 (interspace distance 15)
7. 10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nMDNA 2 (interspace distance 17)
8. 10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nMDNA 2 (interspace distance 20)
9. 10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 2 (interspace distance 22)
10. 10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25)
11. 10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nM DNA 3 (interspace distance 27)
12. 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.

BRET

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.

G-block 2 and 3 were also digested for regular ligation. However, this had failed due to too low concentrations.

Peking

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.

Split-NL

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:

1. 10 nM Sbit + 10 nM Lbit
2. 10 nM Sbit + 10 nM Lbit + 1.2 nM DNA 1
3. 10 nM Sbit + 10 nM Lbit + gRNA 3 + 1.2 nM non-target DNA
4. 10 nM Sbit + 10 nM Lbit + gRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
5. 10 nM Sbit + 10 nM Lbit + gRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
6. 10 nM Sbit + 10 nM Lbit + gRNA 3 + 1.2 nM DNA 1 (interspace distance 15)

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:

1. 2 nM Sbit + 2 nM Lbit
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM DNA 1
3. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM non-target DNA
4. 2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
5. 2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
6. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 1 (interspace distance 15)
7. 2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 2 (interspace distance 17)
8. 2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 2 (interspace distance 20)
9. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 2 (interspace distance 22)
10. 2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 3 (interspace distance 25)
11. 2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 3 (interspace distance 27)
12. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 3 (interspace distance 30)

BRET

The Gibson Assembly mixture was transformed into XL10-Gold competent cells.

BRET

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.

BRET

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.

Moreover, restriction digestions were performed to form a new set of digested gBlocks, which were subsequently purified by agarose gel.

Characterization

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 2016 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.

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.

BRET

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.

DNA was extracted from yesterday's gel slices containing the relevant digested parts (/SacI/-BRETp2-/NheI/; /NheI/-BRETp3-/XhoI/; /AgeI/-pET28a(+)-dCas9-Lbit-/SacI/) using the Qiagen gel extraction kit. With these, new ligation reactions were performed for ligating /SacI/-BRETp2-/NheI/ in /NheI/-pET28a(+)-dCas9-Lbit-/SacI/ as well as /NheI/-BRETp3-/XhoI/ in /XhoI/-pET28a(+)-dCas9-Lbit-/NheI/ prepared previously.

Characterization

CT52-mNeonGreen

Transformation was successful for both mutant forms of CT52. Small cultures were made using kanamycin and LB medium.

NanoLuc

The vector for NanoLuc did not contain enough DNA, so we first need to replicate the plasmid DNA. Therefore, transformation in NovaBlue was performed.

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.

1. 2 nM Sbit + 2 nM Lbit | 5mM DTT present in PBS
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM target DNA | 5 mM DTT present in PBS
3. 2 nM Sbit + 2 nM Lbit + sgRNA + non-target DNA |5 mM DTT present in PBS
4. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 10 |5 mM DTT present in PBS
5. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 17 | 5 mM DTT present in PBS
6. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 25 | 5 mM DTT present in PBS
7. 2 nM Sbit + 2 nM Lbit | no DTT
8. 2 nM Sbit + 2 nM Lbit + 1.2 nM target DNA | no DTT
9. 2 nM Sbit + 2 nM Lbit + sgRNA + non-target DNA | no DTT
10. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 10 | no DTT
11. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 17 | no DTT
12. 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.

Split-NL

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.

BRET

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.

Characterization

NanoLuc

The transformation was successful.

mNeonGreen

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.

Split-NL

Sequencing was not successful last week, so new sequencing samples were made.

In addition, additional tests were performed. A new buffer was used containing 20 mM Tris-HCl, 100 mM KCl, 5 mM MgCl₂and 5% glycerol. 30 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.

1. 2 nM Sbit + 2 nM Lbit | Proteins from 22-08
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM DNA 1 | Proteins from 22-08
3. 2 nM Sbit + 2 nM Lbit + sgRNA 1 | Proteins from 22-08
4. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | Proteins from 22-08
5. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 1 | Proteins from 22-08
6. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 2| Proteins from 22-08
7. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 3 | Proteins from 22-08
8. 2 nM Sbit + 2 nM Lbit | Proteins from 27-08
9. 2 nM Sbit + 2 nM Lbit + DNA 1| Proteins from 27-08
10. 2 nM Sbit + 2 nM Lbit + sgRNA 1 | Proteins from 27-08
11. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA| Proteins from 27-08
12. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 1 | Proteins from 27-08
13. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 2 | Proteins from 27-08
14. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 3 | Proteins from 27-08

BRET

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.

Characterization

mNeonGreen

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.

Characterization

Small cultures were made for NanoLuc.

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.

1. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) | sgRNA incubation at 37 °C
2. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) | sgRNA incubation at 37 °C
3. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) | sgRNA incubation at 37 °C
4. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | sgRNA incubation at 37 °C
5. 2 nM Sbit + 2 nM Lbit + sgRNA 1 | sgRNA incubation at 37 °C
6. 2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA
7. 2 nM Sbit + 2 nM Lbit
8. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) | sgRNA incubation at 50 °C
9. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) | sgRNA incubation at 50 °C
10. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) | sgRNA incubation at 50 °C
11. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | sgRNA incubation at 50 °C
12. 2 nM Sbit + 2 nM Lbit + sgRNA 1 | sgRNA incubation at 50 °C
13. 2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA
14. 2 nM Sbit + 2 nM Lbit
15. 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.

Split-NL

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.

1. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 3 (interspace distance 30)
2. 2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 3 (interspace distance 27)
3. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25)
4. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 2 (interspace distance 22)
5. 2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 2 (interspace distance 20)
6. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17)
7. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 1 (interspace distance 15)
8. 2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
9. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
10. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA
11. 2 nM Sbit + 2 nM Lbit + sgRNA 1
12. 2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA
13. 2 nM Sbit + 2 nM Lbit

BRET

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.

Characterization

We had a meeting with one of our supervisors and we decided to characterize mCherry.

Split-NL

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.

1. 2 nM Sbit + 2 nM Lbit | 5 min centrifugation after incubation
2. 2 nM Sbit + 2 nM Lbit + 1.2 non-target DNA | 5 min centrifugation after incubation
3. 2 nM Sbit + 2 nM Lbit + sgRNA 3| 5 min centrifugation after incubation
4. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM non-target DNA | 5 min centrifugation after incubation
5. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + DNA 3 | 5 min centrifugation after incubation
6. 2 nM Sbit + 2 nM Lbit
7. 2 nM Sbit + 2 nM Lbit + 1.2 non-target DNA
8. 2 nM Sbit + 2 nM Lbit + sgRNA 3
9. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM non-target DNA
10. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + DNA 3

Characterization preparation

Cysteine free NanoLuc

Transformation in BL21 was done.

Characterization preparation

Cysteine free NanoLuc

Colonies were present, so small cultures were made.

mCherry

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.

Split-NL

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.

1. 2 nM Sbit + 2 nM Lbit
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA
3. 2 nM Sbit + 2 nM Lbit + gRNA
4. 2 nM Sbit + 2 nM Lbit + gRNA + 1.2 nM nontarget DNA
5. 2 nM Sbit + 2 nM Lbit + gRNA + 1.2 nM DNA 3 (interspace distance 30)
6. 1 nM Sbit + 1 nM Lbit
7. 1 nM Sbit + 1 nM Lbit + 1.2 nM nontarget DNA
8. 1 nM Sbit + 1 nM Lbit + gRNA
9. 1 nM Sbit + 1 nM Lbit + gRNA + 1.2 nM nontarget DNA
10. 1 nM Sbit + 1 nM Lbit + gRNA + 1.2 nm DNA 3 (interspace distance 30)
11. 0.5 nM Sbit + 0.5 nM Lbit
12. 0.5 nM Sbit + 0.5 nM Lbit + 1.2 nM nontarget DNA
13. 0.5 nM Sbit + 0.5 nM Lbit + gRNA
14. 0.5 nM Sbit + 0.5 nM Lbit + gRNA + 1.2 nM nontarget DNA
15. 0.5 nM Sbit + 0.5 nM Lbit + gRNA + 1.2 nM DNA 3 (interspace distance 30)

Characterization

mCherry & Cysteine-free NanoLuc

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 18 °C at 160 rpm.

Split-NL

We performed tests using variable concentrations of mNeonGreen-NanoLuc to determine which concentration we would be using in our experiments later on. 30 μL samples were used in the Tris-HCl, MgCl₂buffer containing DTT and BSA. NanoGlo was added in 2000X dilution.

1. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM DNA 3 (interspace distance 30)
2. 0.1 pM mNeonGreen-NanoLuc
3. 0.2 pM mNeonGreen-NanoLuc
4. 1 pM mNeonGreen-NanoLuc
5. 2 pM mNeonGreen-NanoLuc
6. 5 pM mNeonGreen-NanoLuc
7. 10 pM mNeonGreen-NanoLuc
8. 20 pM mNeonGreen-NanoLuc

mCherry

The large culture of mCherry was purified with Nickle affinity chromatography.

Fluorescence was measured.

• 96 Well plate
• A7. 1nM mCherry
• A8. 1pM mCherry
• A9. 10 pM mCherry
• A10. 100 pM mCherry
• A11. 10 nM mCherry

Cysteine free NanoLuc

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.

Phage experiments

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 10¹¹PFU/mL was also taken back to Eindhoven to denature by heat and allow for testing our split-NL sensor on whole T7 genome.

Split-NL

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 6x 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.

1. 2 nM Sbit + 2 nM Lbit
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA
3. 2 nM Sbit + 2 nM Lbit + gRNA
4. 2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA + gRNA
5. 2 nM Sbit + 2 nM Lbit + gRNA 1 + 0.1 nM T7 phage DNA (interspace distance 17)
6. 2 nM Sbit + 2 nM Lbit + gRNA 2 + 0.1 nM T7 phage DNA (interspace distance 20)
7. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 0.1 nM T7 phage DNA (interspace distance 22)

BRET

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:

• One complete backbone was made (A, ca. 5.3 kb)
• 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).

Afterwards, the resulting PCR mixtures were PCR purified and samples were run on an agarose gel to check for correct PCR products.

mCherry & Cysteine free NanoLuc

The samples from the purification were put on an SDS-PAGE gel.

BRET

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.

mCherry

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 30 min UV light exposure was also measured.

Phage experiments at QAMH Brussels

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 E. coli B 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 E coli B 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.

Split-NL

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.

BRET

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.

Phage experiments at QAMH Brussels

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. OD₆₀₀ 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 FeSO₄, 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.

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:

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:

1. 0.0 g/L Glucose
2. 0.01 g/L Glucose
3. 0.25 g/L Glucose
4. 0.5 g/L Glucose
5. 1.0 g/L Glucose
6. 2.0 g/L Glucose
7. 4.0 g/L Glucose
8. 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.

Split-NL

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.

BRET

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.

Phage experiments at QAMH Brussels

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.

We also performed a first trial of the experiment for determination of the bacteriophage adsorption rate. A dilution of a mid-log phase culture (OD₆₀₀ 0.549) was made (resulting in OD₆₀₀ 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^-7 and 10^-6 of the 3∙10¹¹ stock. The resulting solutions were plated and incubated overnight.

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 OD₆₀₀to CFU/ml reliably.

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:

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.

Split-NL

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 10 pM were used and the same incubation conditions were used as in previous tests.

BRET

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.

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.

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.

Split-NL

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.

Phage experiments at QAMH Brussels

A successfully grown overnight culture was used to repeat the phage adsorption rate determination experiment, with a start OD₆₀₀ of 0.308 and phage titer 7∙10⁴. 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.

We also took pictures of all our plates that we accumulated thus far, one examples clearly showing plaques is shown below.

Figure 6: One of the accumulated plates.

Split-NL

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.

Phage experiments at QAMH Brussels

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.

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).

The adsorption rate determination experiment was performed one last time, over a period of 20 minutes this time. Starting OD₆₀₀was 0.096, with an OD₆₀₀ 0.142 reached 30 minutes after finishing a sampling period of 20 minutes. Note: The origin culture grew remarkably well this time, obtaining an OD₆₀₀ 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 OD₆₀₀ values to CFU/ml.

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 OD₆₀₀ values.

Split-NL

Three different bioluminescence tests were performed: variable interspace distance, a range of phage DNA concentrations and a calibration test using NanoLuc-mNeonGreen.

Variable interspace distance

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.

Range of phage DNA concentrations

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.

Calibration test using NanoLuc-mNeonGreen

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.

Split-NL

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.