Week 21 (20th-26th of May)

22nd of May

1. Construct and primer design

Team members: Ojas and Hitesh

Used Snapgene software to design the USER-primers for the gene constructs while the genes were codon optimised online in IDT and ordered as it is.

Materials

Gene sequences
Snapgene software

Procedure

A special attention was given to avoid the restriction enzyme recognition site
1. for USER NotI
2. for 3A assembly system i.e. EcoRI, SpeI, XbaI and PstI
3. For Golden gate assembly SapI
We also did a tag for myc-tag in the C-terminus of all the receptors in order to keep other methods of characterization open.
We didn’t design the primers for linker-sfGFP sequence nor did we order the sequence, we are going to use the already available resource in the lab (Karel, Nat, Lucas etc)

2. Dilution of primers and gene fragments

Team members: Claudia, Ojas and Hitesh

Dilute the gene fragments and primers

Materials

Milli-Q water
Gene fragments: Human LHCGR, XLHCGR and GPER
Primers: pCCW12-F, pCCW12-R, XLHCGR-F, XLHCGR-R, XLHCGR-linker-R, sfGFP-linker-F, sfGFP-R, GPER-F, GPER-R, GPER-linker-R

Procedure

For primer dilution
- Took the samples delivered by the IDT in powder form to dilute in Milli-Q-water and make a final stock solution to 100 μM. To do that, the OD values were given in nM concentrations and amount of water to be added to make a final concentration of 100 μM was calculated as in Table 1.
- Further took a part of the Stock solution to make a working solution of 10 μM. To do so, took 10 μL of each stock solution and added 90 μL of water to it.
For gene fragment dilution
- The gene fragments supplied were 1000 ng each, we added 100 μL of Milli-Q-water to each sample (Human LHCGR, XLHCGR and GPER) to make a final concentration of 10ng/μL

Data

Table 1 The list of primers and their concentration as supplied by IDT and the amount of water to be added to make a final concentration for the stock solution.
Primer Sample name	OD value (nM)	μL of water added
pCCW12-F	28.5	285
pCCW12-R	33.8	338
XLHCGR-F	34.5	345
XLHCGR-R	38.7	387
XLHCGR-linker-R	36.8	368
sfGFP-linker-F	32.2	322
sfGFP-R	29.6	296
GPER-F	37.8	378
GPER-R	33.2	332
GPER-linker-R	35.9	359

Results

Stock solutions stored in the freezer at -20 degrees
Working solution arranged to be used also at freezer

3. PCR Amplification: X7-PFU Polymerase

To amplify the gene sequences and other promoter regions.

Materials

10X X7 PCR buffer
dNTP
F-primers (10 μM)
R-primers (10 μM)
Template
X7-Polymerase
mQ Water
Samples: XLHCGR, GPER, GPA1-Gαi, GPA1-Gαs
pCCW12

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

Data

Table 2 The composition of materials to be used per PCR amplification sample
Materials	Quantity (μL)
10X X7 PCR buffer	5
dNTPs	4
F-primer (10 μM)	2.5
R-primer (10 μM)	2.5
Template	0.5
X7-Polymerase	0.5
mQ Water	?
Total	50

Genes

SERIAL NO.	NUCLEOTIDE FRAGMENT	F-PRIMER	R-PRIMER
1	GPER	GPER-F	GPER-R
2	GPER-linker	GPER-F	GPER-linker-R
3	XLHCGR	XLHCGR-F	XLHCGR-R
4	XLHCGR-linker	XLHCGR-F	XLHCGR-linker-R
5	sfGFP	sfGFP-F	sfGFP-R
6	GPA1-Gαi	GPA1-F	GPA1-Gαi-R
7	GPA1-Gαs	GPA1-F	GPA1-Gαs-R
8	STE12	STE12-F	STE12-R
9	ZsGreen	ZsGreen-F	ZsGreen-R

Promoters


SERIAL NO.	PROMOTER FRAGMENT	F-PRIMER	R-PRIMER	Specific for gene
10	pCCW12	pCCW12-F	pCCW12-R	Receptor
11	pPGK1	pPGK1-F	pPGK1-R	Gαs and Gαi
12	pRET2	pRET2-F	pRET2-R	STE12
13	pFIG1	pFIG1-F	pFIG1-R	Reporter

Results

In the experimental setup, the amplified sample numbers were labelled such and were arranged serially for further gel electrophoresis.

SAMPLE NO.	NUCLEOTIDE FRAGMENT
1	GPER
2	XLHCGR
3	pCCW12
4	GPA1-Gαi
5	GPA1-Gαs
6	pPGK1
7	STE12
8	pRET2
9	ZsGreen
10	pFIG1
11	GPER-linker
12	XLHCGR-linker
13	sfGFP
14	Water: negative control

Conclusion

All the amplified fragments were achieved, this was then to be confirmed for correct amplification by running gel electrophoresis.

23rd of May

1. GEL ELECTROPHORESIS

Team members: Hitesh

To check for the amplified gene sequences and other promoter regions.

Materials

Agarose
1X TAE-buffer
Loading dye
DNA ladder
PCR tubes
Ice box

Procedure

To prepare the gel
1. 1% agarose gel was taken from the common stock in electrophoresis room
2. The Agarose-TAE buffer solution was poured into the casting tray. Please label the cast with iGEM after adding the comb to the solution.
Setting up the electrophoresis chamber
1. Solidified gel was placed on the electrophoresis chamber filled with TAE buffer
2. To prepare the sample to be loaded, mixed 4 μL of each sample was taken into a new PCR tube and 1 μL of loading dye was added to each sample.
3. To load the wells, the first and the last well were filled with the 1 kb DNA ladder while wells 2 to 13 were loaded with the PCR samples (1-13) while 14 was loaded with negative control.
Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 25 mins.

Data


SAMPLE NO.	NUCLEOTIDE FRAGMENT	LENGTHS (Kb)
1	GPER
2	XLHCGR
3	pCCW12
4	GPA1-Gαi
5	GPA1-Gαs
6	pPGK1
7	STE12
8	pRET2
9	ZsGreen
10	pFIG1
11	GPER-linker
12	XLHCGR-linker
13	sfGFP
14	Water: negative control

24th of May

1. NORMAL PCR AMPLIFICATION: X7-PFU POLYMERASE

Team members: Anett, Iben and Hitesh

The same protocol as NORMAL PCR AMPLIFICATION: X7-PFU POLYMERASE (22nd of May) was performed again to amplify the gene sequences and the promoter regions for segments 1, 2, 4 and 9 because the amplification for these failed as confirmed via gel electrophoresis.

Materials

10X X7 PCR buffer
dNTP
F-primers (10 μM)
R-primers (10 μM)
Template: XLHCGR, GPER, GPA1-Gαi, ZsGreen (Sample number 1,2,4 and 9 as mentioned in 3. Normal PCR amplification)
X7-Polymerase
mQ Water

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 with everything 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

Data

**CAPTION MISSING**
Materials	Quantity (μL)
10X X7 PCR buffer	5
dNTPs	4
F-primer (10 μM)	2.5
R-primer (10 μM)	2.5
Template	0.5
X7-Polymerase	0.5
mQ Water	?
Total	50

Results

In the experimental setup, the amplified sample numbers were labelled such and were arranged serially for further gel electrophoresis.

**CAPTION MISSING**
SAMPLE NO.	NUCLEOTIDE FRAGMENT
1	GPER
2	XLHCGR
3	GPA1-Gαi
9	ZsGreen
Negative control	Mix without template

Conclusion

All the amplified fragments were confirmed with gel electrophoresis again.

2. Gel Electrophoresis

Team members: MISSING

The same protocol was performed as in 3.GEL ELECTROPHORESIS (22nd of May). Samples 1, 2 ,4 and 9 were run on a gel along with a negative control.

The gel was loaded in the following order:
Well 1: 1 kb ladder
Well 2: sample 1
Well 3: sample 1
Well 4: sample 2
Well 5: sample 2
Well 5: sample 4
Well 6: sample 4
Well 7: sample 9
Well 8: sample 9
Well 9: negative control
Well 10: 1 kb ladder

SAMPLE NO.	NUCLEOTIDE FRAGMENT
1	GPER
2	XLHCGR
3	GPA1-Gαi
9	ZsGreen
Negative control	Mix without template

3. Purification of Amplified Product

Team members: Swenja, Selma, Signe and Hitesh

This step was carried out to purify the DNA fragments from the 13 samples of PCR amplification product directly instead of digested PCR product (with USER enzymes). Usually Gel extraction is carried out for purification, which results in loss of DNA. So, a PCR clean up kit was used.

Materials

Omega PCR clean up kit (Taken from the Gel electrophoresis room). The kit contains: HiBind mini columns, collection tubes, CP buffer, Wash buffer and Elution buffer

Procedure

40 microlitres of all the PCR samples as mentioned in the Table below were transferred into a 1.5 mL microcentrifuge tube.
200 microlitres of the CP buffer was added.
The samples were vortexed and centrifuged briefly.
HiBind DNA mini columns were inserted into a 2 mL collection tube.
The samples from step 3 were added to the HiBind DNA mini column.
Centrifugation was carried out at 13000 g for 60 seconds at room temperature. The filtrate was discarded and the collection tubes were reused.
700 microlitres of DNA wash buffer diluted with 100% ethanol was added. Centrifugation was carried out at maximum speed for 60 seconds. The filtrate was discarded and the collection tubes were reused.
Step 7 was repeated again for a second DNA wash buffer step.
The empty HiBind DNA mini columns were centrifuged at maximum speed for 2 minutes to dry the column. This step was critical to remove the trace ethanol.
The HiBind DNA mini column was transferred into a new 1.5 mL microcentrifuge tube.
50 microlitres of elution buffer was directly added to the center of the column matrix. It was kept at room temperature for 2 minutes and was centrifuged at maximum speed for 60 seconds.
The DNA was stored at -20⁰C.

Data

SAMPLE NO.	NUCLEOTIDE FRAGMENT
1	GPER
2	XLHCGR
3	pCCW12
4	GPA1-Gαi
5	GPA1-Gαs
6	pPGK1
7	STE12
8	pRET2
9	ZsGreen
10	pFIG1
11	GPER-linker
12	XLHCGR-linker
13	sfGFP

Results

Purified samples were stored in the freezer at -20 °C. Samples 1-13 are still in the freezer in the eppendorf tube stand labelled ‘Purified DNA 24/05/2019’