Week 33 (12th-18th of August)

12th August 2019

1. USER LIGATION

Team members: Anett and Noël

We repeated the USER ligation of TPSTII.

Procedure

We mixed the following solutions in a PCR tube:


pTDH	1.5 µL
TPST I	1 µL
TPST II	1 µL
10x USER enzyme	1 µL
10x CutSmart buffer	1 µL
Water	3.5 µL

We used the PCR machine for ligation with the program: iGEM USER: 37°C for 30 min, 25°C for 15 min, 20°C for 10 min.

2. E. COLI TRANSFORMATION

Team members: Swenja and Noël

E. coli transformation was performed for the following samples:


Sample nr.	Construct	Date of creation
1	Ass2C + pTDH + TPSTI	08/08
2	Ass2C + pTDH + TPSTII	12/08
3	X3A + pCCW12 + HuLHCGR + li + sfGFP	08/08
4	X3A + pCCW12 + HuLHCGR	08/08
5	X3A + pCCW12 + GPER	08/08
6	X3A + pCCW12 + XLHCGR	08/08
7	X3A + pCCW12 + XLHCGR+ li+ sfGFP	08/08
8	Ass2A + pPGKI + Gαs	08/08
9	Ass2C + pTDH + TPSTI	08/08

Materials

Competent E. coli cells in aliquots of 50 µl
LB-media
LB plates with Carbecillin (500 µl per 250 ml)

Procedure

Competent cells were collected from the freezer (- 80 °C) and thawed on ice. Each of the tubes should contain 50 µl.
The USER ligated samples (approx. 8 µl) were added to the cells.
The samples were kept on ice for 10 mins.
After incubation on ice, the samples were heat shocked for 30-45 s on the heat block at 42 °C and subsequently briefly cooled down.
Under the clean bench, 1 ml of LB media (w/o antibiotic) was added to each tube.
The bacteria were then recovered for 60 min on the heat block at 37 °C.
After incubation the cultures were span down for 1 min at 22,4 °C and 8000 rcf.
900 µl of the supernatant were removed from each tube and the pellet resuspended in the remaining media.
The cultures were then spread on LB plate with Carbenicillin and incubated O/N at 37 °C.

Results

Unfortunately, the plates were unusable as there were to many colonies. As a result no single colonies could be picked and both USER ligation and E. coli transformation have to be repeated. The reason for that is, that too much sample has been used for the transformation as well as the whole culture spread on one plate, respectively. Next time, only 4 µl of the samples should be used to yield less colonies and to allow a repetition of the E. coli transformation without having to redo USER ligation. Furthermore, only around 75 µl should be spread on each plate.

13th August 2019

1. REPETITION OF USER LIGATION

Team members: Claudia and Signe

We did USER ligation on the same samples as on the 8th of August as the plates had too many colonies for us to do colony PCR./p>

The USER ligation was done as according to the protocol from the 8th of August.

Procedure

To the samples with the linker sfGFP was also added. We added 1 µL sfGFP.

2. REPETITION OF E. COLI TRANSFORMATION

Team members: Anett and Swenja

E. coli transformation was repeated for the following samples:


Sample nr.	Construct	Date of creation
1	Ass2C + pTDH + TPSTI	13/08
2	Ass2C + pTDH + TPSTII	13/08
3	X3A + pCCW12 + HuLHCGR + li + sfGFP	13/08
4	X3A + pCCW12 + HuLHCGR	13/08
5	X3A + pCCW12 + GPER	13/08
6	X3A + pCCW12 + XLHCGR	13/08
7	X3A + pCCW12 + XLHCGR+ li+ sfGFP	13/08
8	Ass2A + pPGKI + Gαs	13/08
9	Ass2C + pTDH + TPSTI	13/08

Material

Competent E. coli cells in aliquots of 50 µl
LB-media
B plates with Carbecillin (500 µl per 250 ml)

Procedure

Competent cells were collected from the freezer (- 80 °C) and thawed on ice. Each of the tubes should contain 50 µl.
4 µl of the USER ligated samples were added to the cells.
The samples were kept on ice for 10 mins.
After incubation on ice, the samples were heat shocked for 30-45 s on the heat block at 42 °C and subsequently briefly cooled down.
Under the clean bench, 1 ml of LB media (w/o antibiotic) was added to each tube.
The bacteria were then recovered for 60 min on the heat block at 37 °C.
After incubation the cultures were span down for 1 min at 22,4 °C and 8000 rcf.
900 µl of the supernatant were removed from each tube and the pellet resuspended in the remaining media.
For each sample, both 100 µl and 50 µl were then spread on two different LB plates with Carbenicillin and incubated O/N at 37 °C.

Results

There was a good growth on all plates with individual colonies.

14th of August

1. COLONY PCR OF YEAST

Team members: Claudia and Hitesh

Colony PCR was performed

Procedure

8 colonies were picked from each plate


Date	Plate name
13/8	GPER+pCCW12+X3A
13/8	XLHCGR+sfGFP+pCCW12+X3A
13/8	XLHCGR+pCCW12+X3A
13/8	Gαs+pPGK1+Ass2C
13/8	TPST1+Ass2C+pTDH3
13/8	TPST2+Ass2C+pTDH3
13/8	HuLHCGR+sfGFP+pCCW12+X3A
13/8	HuLHCGR+pCCW12+X3A

Colony PCR was performed according to the protocol on the 28th of May.

Results

Of the 64 tested colonies none was positive which caused us to suspect an unsuccessful USER ligation.

15th-16th August 2019

1. COLONY PCR OF YEAST

Team members: Benedicte and Ojas

A similar colony PCR was performed on the 14th of August, but due to issues with the PCR machine, the experiment was not concluded and is therefore here repeated. The PCR was performed on the 15th and the gel was run on the 16th.

Procedure

15 colonies were picked from 8 plates to a total of 60 samples.


Date	Plate name
13/08	GPER+pCCW12+X3A
13/08	XLHCGR+sfGFP+pCCW12+X3A
13/08	XLHCGR+pCCW12+X3A
13/08	Gαs+pPGK1+Ass2C
13/08	TPST1+Ass2C+pTDH3
13/08	TPST2+Ass2C+pTDH3
13/08	HuLHCGR+sfGFP+pCCW12+X3A
13/08	HuLHCGR+pCCW12+X3A

A colony PCR as described on the 15th of July 2019 was performed with the following change: 10 µL of the Mastermix were added to 1,5 µL of the sample.