Week 28 (8th-14th of July)

8th of July

1. 2nd PCR amplification: X7-PFU Polymerase and gel electrophoresis

Team members: Claudia & Hitesh

PCR amplification from July 5th were inconclusive, so we aim to repeat the process. To amplify the human LH receptor gene, amplification is performed with the reverse primer with or without a linker to make LH receptor gene.

Materials

10X X7 PCR buffer
dNTP
F-primers (10 μM): HuLHCGR-F
R-primers (10 μM): HuLHCGR-R and HuLHCGR-linker-R
Template
X7-Polymerase
mQ Water
Template: HuLHCGR

Procedure

Noted the number of samples to be PCR amplified and calculated the quantities of each material to be added for the master-mix. It should be noted that the amount of water added is to make the total solution of 50 μL.
The mastermix for PCR included the buffer, dNTPs, water and X7 polymerase. Mixed the total quantity required based on the number of PCR amplification samples.
Then, we pipette out the individual quantities of Master-mix into each labelled PCR tube.
Now added the primers specific to each PCR sample and finally the template specific for each PCR sample.
Gently mix the final sample, vortex before setting up the PCR machine.
Also, prepare a negative control which includes the entire mix except for the template.
Put the samples into the machine and select the program based on the requirement finally start the program.
PCR program 30 cycles of:
1. 98 degree celsius: 30 seconds
2. 98 degree celsius: 20 seconds
3. 55 degree celsius: 25 seconds
4. 72 degree celsius: 2 minutes and 30 seconds
5. 72 degree celsius: 5 minutes
6. 12 degree celsius: Infinity

Results

Wells from the left: 1 is ladder, 2 is NC, 3 is huLHCGR, 4 huLHCGR-linker, 5 is empty, 6 is ladder.

The gel showed several undesired bands and some of them were more clear than the desired. We therefore decided to do yet another PCR to try to optimize the specificity.

2. Sequencing for GPER, GPER-sfGFP, XLHCGR and XLHCGR-sfGFP

Team members: Ojas

We repeated the sequencing from 2nd July, 2019 for the samples 1. X3A-pCCW12-GPER 2. X3A-pCCW12-GPER-sfGFP 3. X3A-pCCW12-XLHCGR-sfGFP 4. X3A-pCCW12-XLHCGR that we extracted on 1st July 2019 were prepared for sequencing.

Materials

Samples that were prepared July 1st 2019 as mentioned in the table below also.
Primers
Water

Procedure

The concentration of each purified plasmid DNA sample was measured using NanoDrop.
A stock solution of the primers were used from the freezer after usage.
To assure that the whole constructs would be synthesised 3 to 4 primers were used for each vector (USER primer of the first gene and two further primers according to list in R148) Note: We used Sanger sequencing which is limited to the sequencing of approximately 700 bp at a time. Therefore an extra primer was used.
For each construct only one sample was prepared for sequencing (Samples 1.1, 1.2, 2.2, 2.3, 3.1, 3.2, 4.1 and 4.2). Moreover the overview of samples prepared for sequencing is mentioned in the table The samples were prepared as follows:
1. DNA: µl corresponding to 500 ng DNA
2. Primer: 3,3 µl
3. mH20: x µl
4. Reaching a total of 10 µl
Stickers with QR codes. The stickers consists of two parts, the smaller of which was attached into the lab book in a list next to the corresponding construct and primer. The second part was wrapped around the eppendorf tubes in such a way that the QR code was clearly visible.

Data

The E coli colonies transformed with these respective vectors were in the plates labelled 1. X3A-pCCW12-GPER 2. X3A-pCCW12-GPER-sfGFP 3. X3A-pCCW12-XLHCGR-sfGFP 4. X3A-pCCW12-XLHCGR and the positively transformed colonies were grown in liquid LB medium overnight and labelled on the basis of the plate and the colony number as mentioned in the table with plate number following with a decimal and then the colony number.
Name of the Insert	Our name of the samples (corresponds to number of colony on the plate the liquid culture was inoculated with)
X3A-pCCW12-GPER	1.1 and 1.2
X3A-pCCW12-GPER-sfGFP	2.2 and 2.3
X3A-pCCW12-XLHCGR-sfGFP	3.1 and 3.2
X3A-pCCW12-XLHCGR	4.1 and 4.2

Table shows nanodrop concentration for each sample and the calculated volume of template that needs to be added for sequencing in each sample.
Construct	Sample Nr.	A60/280		A260/230		ng/µl		Avg ng/uL	µl used
X3A-pCCW12-GPER	1.1	1.81	1.84	1.76	2.02	172.1	156.8	164.45	3.04
X3A-pCCW12-GPER	1.2	1.83	1.81	2.06	1.86	168.3	182.0	175.15	2.85
X3A-pCCW12-GPER-sfGFP	2.2	1.80	1.86	1.72	2.09	224.9	214.4	219.65	2.28
X3A-pCCW12-GPER-sfGFP	2.3	1.83	1.85	1.79	2.08	241.3	232.0	236.65	2.11
X3A-pCCW12-XLHCGR-sfGFP	3.1	1.84	1.87	2.28	2.24	207.3	202.2	204.7	2.44
X3A-pCCW12-XLHCGR-sfGFP	3.2	1.83	1.86	2.17	2.22	211.1	203.5	207.3	2.41
X3A-pCCW12-XLHCGR	4.1	1.82	1.85	1.95	2.22	180.0	168.5	174.25	2.87
X3A-pCCW12-XLHCGR	4.2	1.79	1.79	1.79	1.79	203.7	203.5	203.6	2.46

Overview of the sample preparation
Sample name (see “36 dated 1st July 2019”)	Primer 1	Primer 2	Primer 3	Primer 4	Used Primers
1.1 and 1.2	YEA74	GPER-F		YEA81	Y81, Y74, GPER-F
2.2 and 2.3	YEA74	GPER-F	GPER-Li-R	YEA81	Y81, Y74, GPER-F, GPER-R
3.1 and 3.2	YEA74	XLHCGR-F	XLHCGR-Li-R	YEA81	Y81, Y74, XLH-F, XLH-R
4.1 and 4.2	YEA74	XLHCGR-F		YEA81	Y81, Y74, XLH-F

Results


Construct	Primers+Sequencing label	Analysis Individual Fragment	Analysis Plasmid level
1.1 X3A-pCCW12-GPER	YEA81: 1CB8ZAB038	The sequencing data file aligned just perfectly to the template. At 429 a T-A base pair is not properly sequenced.	The sequencing data file aligned just perfectly to the template. At 7542 a T-A base pair is not properly sequenced.
	USER-GPER-F: 1CB8ZAB037	The sequencing data file aligned just perfectly to the template. At 21 bp a T-A bp is poorly sequenced but overlapping region of good quality confirmed it is correct.
	YEA74: 1CB8ZAB036	The sequencing data file aligned just perfectly to the template. The sequencing data started to deteriorate after 1070 bp.
1.2 X3A-pCCW12-GPER	YEA81: 1CB8ZAB041	The sequencing data file aligned just perfectly to the template. At 431 bp and 432 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.	The sequencing data file aligned just perfectly to the template. At 7541 bp and 7542 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.
	USER-GPER-F: 1CB8ZAB040	The sequencing data file aligned just perfectly to the template.
	YEA74: 1CB8ZAB039	The sequencing data file aligned just perfectly to the template.
2.2 X3A-pCCW12-GPER-Li-sfGFP	YEA74: 1CB8ZAB042	The sequencing data file aligned just perfectly to the template. At 429 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.	The sequencing data file aligned just perfectly to the template. At 8280 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.
	USER-GPER-F: 1CB8ZAB043	The sequencing data file aligned just perfectly to the template.
	USER-GPER-Li-R: 1CB8ZAB044	The sequencing data file aligned just perfectly to the template. Although we did find a few mismatches at 891bp (A to G) and 1049 (G to A) but the quality of these regions were low compared to other overlapping regions sequenced by another primer. Therefore, it was assumed to be a perfect sequence.
	YEA81: 1CB8ZAB045	The sequencing data file aligned just perfectly to the template.
2.3 X3A-pCCW12-GPER-sfGFP	YEA74: 1CB8ZAB046	The sequencing data file aligned just perfectly to the template.	The sequencing data file aligned just perfectly to the template. At 8280 bp, the region is rich in T-A bases therefore the sequencing data quality is poor. Substitution G-A at 6789 bp
	USER-GPER-F: 1CB8ZAB047	The sequencing data file aligned just perfectly to the template.
	USER-GPER-Li-R: 1CB8ZAB048	Substitution G-A at 31bp
	YEA81: 1CB8ZAB049	The sequencing data file aligned just perfectly to the template. At 427 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.
3.1 X3A-pCCW12-XLHCGR-Li-sfGFP	YEA74: 1CB8ZAB022	The sequencing data file aligned just perfectly to the template. At 431 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.	The sequencing data file aligned just perfectly to the template. At 9297 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.
	USER-XLHCGR-F: 1CB8ZAB023	It is poorly sequenced data. At 91 bp, the region is poorly sequenced but overlapping sequencing data covered by YEA74 confirmed that the sequence is correct. At 261 bp the region is poorly sequenced	It is poorly sequenced data. At 8785 bp, the region is poorly sequenced but overlapping sequencing data covered by YEA74 confirmed that the sequence is correct. At 8614 bp the region is poorly sequenced
	USER-XLHCGR-Li-R: 1CB8ZAB024	The sequencing data file aligned just perfectly to the template upto 1120 bp from primer amplification site	The sequencing data file aligned just perfectly to the template from 6787 bp upto 7883 bp
	YEA81: 1CB8ZAB025	At 582 an A-T changed to G-C causing Leucine to Proline change in one amino acid only.	At 6526 an A-T changed to G-C causing Leucine to Proline change in one amino acid only.
3.2 X3A-pCCW12-XLHCGR-Li-sfGFP	YEA74: 1CB8ZAB026	The sequencing data file aligned just perfectly to the template. At 431 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.	The sequencing data file aligned just perfectly to the template. At 9297 bp, the region is rich in T-A bases therefore the sequencing data quality is poor.
	USER-XLHCGR-F: 1CB8ZAB027	It is poorly sequenced data.	It is poorly sequenced data.
	USER-XLHCGR-Li-R: 1CB8ZAB028	The sequencing data file aligned just perfectly to the template.	The sequencing data file aligned just perfectly to the template.
	YEA81: 1CB8ZAB029	The sequencing data file aligned just perfectly to the template.	The sequencing data file aligned just perfectly to the template.
4.1 X3A-pCCW12-XLHCGR	YEA74: 1CB8ZAB030	The sequencing data file aligned just perfectly to the template.	The sequencing data file aligned just perfectly to the template.
	USER-XLHCGR-F: 1CB8ZAB031	Poor sequencing data	Poor sequencing data
	YEA81: 1CB8ZAB032	The sequencing data file aligned just perfectly to the template.	The sequencing data file aligned just perfectly to the template.
4.2 X3A-pCCW12-XLHCGR	YEA74: 1CB8ZAB033	The sequencing data file aligned just perfectly to the template.	The sequencing data file aligned just perfectly to the template.
	USER-XLHCGR-F: 1CB8ZAB034	No sequencing data	No sequencing data
	YEA81: 1CB8ZAB035	The sequencing data file aligned just perfectly to the template.	The sequencing data file aligned just perfectly to the template.

9th of July

1. 3rd PCR amplification: X7-PFU Polymerase and gel electrophoresis

Team members: Benedicte & Mads

As on the 5th and 8th of July we struggled to get proper specificity in the PCR amplification. We tried again to amplify the human LH receptor gene. Amplification is performed with the reverse primer with or without a linker to make LH receptor genes with or without linker.

Results

Wells from the left: 1 is ladder, 2 is negative control, 3 is huLHR and 4 is huLHR-linker, 5 is ladder.

Unfortunately, as is seen in the gel image, we added sample to the negative control (that also included primers), which explains the signal that is seen in negative control. The strongest signals around 2Kbp actually corresponds to the expected sizes of the genes, but there are still issues with specificity. We will therefore make another PCR reaction, and if we still have issues, we can isolate DNA from the rest of this amplified sample, by cutting out this band in the gel.

2. 4th PCR amplification: X7-PFU Polymerase and gel electrophoresis

Team members: Jonas & Iben

The PCR amplification of the human LH receptor gene has failed several times. We aim to redo the amplification, increasing the annealing temperature to get a better yield. Amplification is performed with the reverse primer with or without a linker to make LH receptor genes with or without linker.

Materials and Procedure

The same protocol as the previous days was followed (protocol from July 5th), only changing the annealing temperature during PCR from 55°C → 60°C
Samples were loaded to the gel in the following order:
1. Hu-LHCGR
2. Hu-LHCGR-linker
3. Negative control (no template)

Results

The strong bands are the PCR product we’re looking for, however the polymerase is very inspecific, i.e. the other bands. We will try at a later point to use a different buffer (High Fidelity buffer) which can increase the specificity of the reaction.

11th of July

1. Yeast transformation

Team members: Mads, Jonas and Ojas Materials

O/N culture
YPD media
Cuvettes and spectrophotometer for OD value check
Falcon tubes
Plasmid samples from 1st of july (All modules)
Plasmid samples from 3rd of july (All modules)
NotI enzyme
CutSmart Buffer

Procedure

The O/N culture is diluted 1:10 (100 µl culture + 900 µl YPD media only)
OD of the dilution is measured at 600 nm with pure YPD as standard
1. OD measurement in room R251: OD=0,655. Since there is a linear correlation between the diluted and undiluted O/N culture it is calculated that the OD of the undiluted O/N is 6,55.
The undiluted ON culture is now diluted to achieve an OD of 0,25 for a total volume of 25 mL based on calculation on the OD of the diluted ON culture
1. Calculations: C1*V1=C2*V2 V1=(0,25*25 ml)/6,55=0,9541 ml≈0,95 ml
2. Therefore, Dilution: 0,95 ml of ON culture + 24,05 ml of YPD was prepared
The 25 ml ON dilution (OD 0.25) was then kept in an incubator at 30 °C and 150 RPM for approximately 4 hours. Note the time when you put the yeast into the incubatorm, keep a track of that. (the idea is that the O/N culture of yeast reaches a saturation point where almost all cells are at the same phase and now we prepare a required dilution let the yeast undergone 2 fresh cell divisions)
Prepare the GMO Yeast lab bench at room T-155 in advance and book it on the schedule by mentioning iGEM.
During the 4 hour incubation we prepared the plates (for transformed yeast plating) with GLU -U media (Glucose medium without uracil which would be the selection marker). They were prepared in a sterile bench using already prepared media (plates should be thicker than the plates used to grow E. coli).

At the same time we had to do the ligation of different modules that are in different plasmids in order to transform the yeast with desired module only. To do so, the concentration, volume and amount of DNA was calculated for all the DNA samples which was going to be used. Based on the volume required for yeast transformation and the volume of samples left with us we had to borrow certain samples from Lucas which are also mentioned in the table below.


Sample (from Lucas)	Concentration (ng/µl)
X3C	84.7
BAss2	244
Ass2C	144
Ass2A-pPGK1-GPAGαs	237.6
Ass2B-pRET2-STE12	139
X3C-pFIG1-ZsGreen	244

Table stating the samples we have and their concentrations and volumes.
Sample (1st of july)	Concentration (ng/µl)	Volume left (µl)	ng of DNA
1.1 X3A-pCCW12-GPER	164,45	33	5426,85
2.2 X3A-pCCW12-GPER-sfGFP	219,65	33	7248,45
3.1 X3A-pCCW12-XLHCGR-sfGFP	204,75	30	6142,5
4.1 X3A-pCCW12-XLHCGR	174,25	29	5053,25

Table stating the samples we have and their concentrations and volumes.
Sample (3rd of july)	Concentration (ng/µl)	Volume (µl)	ng of DNA
Ass2A-pGK1-Gαi	79,6	22	1751,2
Ass2B-pRET2-STE12	123,4	22	2717,8
X3C-pFIG1-ZsGreen	150,1	27	4052,7

The uL of DNA we would require were calculated based on the assembler system we are using that is for a 5 asssembler system we calculated uL corresponding to 1000 ng of each plasmid sample while for 3-assembler system we calculated uL corresponding to 600 ng of each plasmid sample and the calculated uL are mentioned in the table below.


S. no.	X3A module	uL	Ass2A module	uL	Ass2B module	uL	Ass2C module	uL	X3C module	uL
1	X3A-pCCW12-GPER	6.1	Ass2A-pPGK1-GPAGαs	4.2	Ass2B-pRET2-STE12	6	Ass2C	6.9	X3C-pFIG1-ZsGreen	4.1
2	X3A-pCCW12-GPER	6.1	Ass2A-pPGK1-GPAGαi	12.6	Ass2B-pRET2-STE12	8.1	Ass2C	6.9	X3C-pFIG1-ZsGreen	6.7
3	X3A-pCCW12-GPER-sfGFP	2.7	BAss2					2.5	X3C	7.1
4	X3A-pCCW12-XLHCGR	5.7	Ass2A-pPGK1-GPAGαs	4.2	Ass2B-pRET2-STE12	4	Ass2C	6.9	X3C-pFIG1-ZsGreen	4.1
5	X3A-pCCW12-XLHCGR	5.7	Ass2A-pPGK1-GPAGαi	9.4	Ass2B-pRET2-STE12	8.1	Ass2C	6.9	X3C-pFIG1-ZsGreen	6.7
6	X3A-pCCW12-XLHCGR-sfGFP	2.9	BAss2					2.5	X3C	7.1
7	A2A-Li-sfGFP

The DNA mixes/samples has to be digested by NotI and left in the buffer for ligation. Therefore the total volume of the digestion reactions has to be calculated along with the amount of buffer needed in each reaction. The buffer volume should be 1/10th of the total volume therefore a calculation was made. For ex. Let volume of buffer to be added be x. Now, x/x+Volume of plasmid sample mix should be equal to 1/10. The further calculated volumes are mentioned in the table below. The enzyme is added as the last component:

Sample Total volume (µl) Volume of buffer (µl) Volume of enzyme (Not1) (µl)

1 28,8 3,2 1,5

2 41,9 4,65 1,5

3 13,3 1,47 1

4 26,4 2,9 1,5

5 38,3 4,2 1,5

6 13,5 1,5 1
After the enzyme has been added quickly votex the samples slightly and thereafter spin the samples down briefly.
The digestion samples then have to incubate at 37 °C for 1,5 hours (usually 2 but we were in a hurry)
Inactivate the enzyme at 65 °C for 20 min and thereafter set the next step at 12 °C indefinitely

2. Yeast transformation: afternoon

Team members: Selma and Hitesh Materials

PLI (0,1M Lithium acetate and 45% PEG)
0,1 M Lithium acetate and Water
YPD

Procedure

OD should be around 1 therefore the culture we incubated in the morning is again checked for its OD (so the yeast cells go through 2 cell divisions and will be easier to transform). The protocol to check OD is the following:

Add 900 µl YPD to 100 µl of yeast cells (10 fold dilution)
Add 1mL YPD to a cuvette as a blank sample to calibrate the spectrophotometer (set it to 0)
Add the samples to a cuvette and place it inside the spectrophotometer
Set the wavelength to 600 nm
Some salmon sperm DNA was denatured by putting it on a heat block at 99 °C
The OD was measured to 0,7 which was too low, so the cultures were incubated for 20 min (in the shaking incubator) to reach the desired OD.
When the desired OD was reached, the cultures ∆5 were transferred from the erlenmeyer flasks to Falcon tubes.
These were centrifuged at 4000 rpm for 5 min
The salmon sperm DNA (that will denature by being boiled for at least 5 min) was cooled down quickly by being put on ice so that it would not re-anneal.
The supernatant from the centrifuged Falcon Tubes containing was discarded (this was just poured out)
The pellet was resuspended in 1 mL Lithium acetate (with water)
The resuspension (1 mL) was transferred into an eppendorf tube
These were centrifuged at 8000 rpm for 1 min and the supernatant was discarded (by pipetting).
The pellet was resuspended in 100 µL Lithium acetate (with water) (Tip: resuspend with big pipette to stress the cells less)
30 µL of denatured salmon sperm DNA was put into the eppendorf tubes (with the yeast cells and the Lithium acetate) and this was gently mixed.
The PLI was mixed by being inverted (not shaken because the PLI is hydroscopic)
1 mL of PLI was put into the eppendorf tubes (PLI stabilizes the cells). It was homogenized by pipetting up and down.
The digested and ligated plasmids was transferred to eppendorf tubes.
The eppendorf tubes were labelled 1-8 following the DNA samples. 8 was the negative control.
200 µL of the mix in eppendorf tubes with the PLI and Lithium acetate and the yeast cells was aliquoted in each of the 8 eppendorf tubes.
The samples were heat shocked at 42 °C for 30 min at 750 rpm
After being heat shocked, the samples were centrifuged for 1 min at 8000 rpm and the supernatant was discarded.
1 mL water was added (to wash the cells) and the cells were resuspended by being vortexed (vortexing is faster and less rough on the cells)
They were again centrifuged for 1 min at 8000 rpm and 900 µL of the supernatant was discarded
The cells were resuspended in the remaining 100 µL and they were mixed gently
They were plated (and named according to the DNA samples) and kept in an incubator for 3 days so colonies could grow

Results

Multiple colonies were observed in all our samples (plates 1-6) No colonies in negative control (plate 8) No colonies in positive control (plate 7), which is a bit strange.

12th July

1. PCR amplification

Team members: Selma and Claudia

PCR amplification was done on the following samples:

pCCwI2
pGkI
pRET2
pFIGI
GpAI-Gαi
GpAI-Gαs
SteI2
ZsGreen

This was done according to the protocol from the 22. May but 100 µL was made instead of 50µL (we made duplicates) The PCR program from the 24th of May was used. After the PCR reaction was done 0,5 µL DPN I was added to the samples, and the samples were incubated at 37 °C for an hour. This is done to make sure that plasmid DNA from the template doesn’t make it into E. coli.