Team:UCopenhagen/Notebook/Week 32







Week 32 (5th-11th of August)

5th August 2019

1. EZNA PLASMID PURIFICATION

Team members: Iben and Ojas

The plasmid purification was performed on 28 samples (in total) of E. coli transformed with different plasmids.

Of which 1-10 samples were from 29th May 2019 while other 2 samples Sample 11 and 12 were from 6th June 2019. These samples from their respective dates were grown on a fresh LB media + Carbenicillin a night before and then used for plasmid purification. However, in the morning 750 uL of each of these E. coli grown in fresh media were stored in eppendorf tubes to be made into glycerol stock later.

Materials

E.Z.N.A. Plasmid DNA Mini Kit I.

Procedure

Followed the protocol as described in the kit entirely as it is.

Data

Sample No. Sample name
1 X3A-pCCW12-GPER
2 X3A-pCCW12-XLHCGR
3 Ass2A-pGK1-Gαi
4 Ass2A-pGK1-Gαi
5 Ass2A-pGK1-Gαs
6 Ass2A-pGK1-Gαs
7 Ass2B-pRET2-STE12
8 Ass2B-pRET2-STE12
9 X3C-pFIG1-ZsGreen
10 X3C-pFIG1-ZsGreen
11 X3A-pCCW12-GPER-sfGFP
12 X3A-pCCW12-GPER-sfGFP

Results

Nanodrop concentrations were measured for the purified plasmids.

Sample No. Sample name Concentrations (ng/uL)
1 X3A-pCCW12-GPER 56.8
2 X3A-pCCW12-XLHCGR 84.1
3 Ass2A-pGK1-Gαi 74.2
4 Ass2A-pGK1-Gαi 61.1
5 Ass2A-pGK1-Gαs 65.2
6 Ass2A-pGK1-Gαs 56.8
7 Ass2B-pRET2-STE12 57.2
8 Ass2B-pRET2-STE12 65.4
9 X3C-pFIG1-ZsGreen 43.7
10 X3C-pFIG1-ZsGreen 46.7
11 X3A-pCCW12-GPER-sfGFP 127.5
12 X3A-pCCW12-GPER-sfGFP 186.7

6th of August 2019

1. YEAST COLONY PCR

Team members: Ojas & Signe

Colony PCR were performed on for the yeast cells transformed on the 1st of August (1, 2, 3 and 5). This was done by using the protocol from 15th of July 2019.

Data

Sample
GPER+Gαi+STE12+Ass2C+ZsGreen (5 assembly)
GPER+Gαs+STE12+Ass2C+ZsGreen (5 assembly)
XLHCGR+Gαi+STE12+Ass2C+ZsGreen (5 assembly)
XLHCGR-sfGFP+B(Ass2)+X3C (3 assembly)

Procedure

  1. 8 colonies were taken from all of the plates (this was done as an advice from the supervisors)
  2. Mastermix - for each sample the following was added: 5 µL of MangoMix , 0.8 µL of each of the three primers (YEA90, YEA85 and YEA89) and 3.5 µL of milli-Q-water to a total volume 10.9 µL.
  3. 1 µL of the corresponding templates were added to the PCR tubes (only for sample 1,2 and 3). 1.5 µL of sample 5 were added.

Results

7th August 2019

1. COLONY PCR OF YEAST

Team members: Claudia and Noël

Procedure

New samples (Red marker: samples 1-8, the black marker dots, also sample 1-8, stems from the first) from the following plates were picked.

Date Plate name
01/08 Glu-U GPER+Gαs 1
01/08 Glu-U GPER+Gαi 2
01/08 Glu-U XLHCGR+Gαs 3
01/08 Glu-U XLHCGR+sfGFP 5

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.

Results

Picture 1: Shows samples red 1-8 from plate 1, and samples red 1-7 from plate 2. All have bands around 1500 bp, which is negative. Red sample 6 from plate 1 shows a very faint band.
Picture 2: Shows samples red 8 from plate 2, samples red 1-8 from plate 3, and samples red 1-6 from plate 5. All have bands around 1500 bp, which is negative. Red sample 5 and 6 from plate 5 show very faint or no bands.
Picture 3: Shows samples red 7 and 8 from plate 5. Sample red 8 from plate 5 shows a faint band at 1500 bp and is negative. Sample red 7 from plate 5 shows a band at 1000 bp which means it is positive.

2. INOCULATION FROM PLATES

Team members: Noel and Claudia

The following yeast colonies were taken from plates and inoculated O/N at 30 °C in to Glu-U media:

Date of plating Plate and colony no. Construct
22/07 2.4 GPER-Gαi
22/07 2.7 GPER-sfGFP
22/07 3.3 XLHCGR-sfGFP
01/08 5.6 XLHCGR-sfGFP

8th August 2019

1. USER LIGATION

Team members: Claudia and Swenja

USER Ligation was performed on the following samples:

Sample no. Nucleotide fragment
1 Ass2C + pTDH + TPSTI
2 Ass2C + pTDH + TPSTII
3 X3A + pCCW12 + HuLHCGR + li + sfGFP
4 X3A + pCCW12 + HuLHCGR
5 X3A + pCCW12 + GPER
6 X3A + pCCW12 + XLHCGR
7 X3A + pCCW12 + XLHCGR+ li+ sfGFP
8 Ass2A + pPGKI + Gαs

Procedure

We mixed the following solutions in a PCR tube:

10x USER enzyme 1 µL
10x Cutsmart buffer 1 µL
Promoter x µL (corresponding to at least 10 ng)
Gene x µL (corresponding to at least 10 ng)
Vector DNA 1 µL/td>
Water x µL
In total 10 µL

A mastermix was prepared consisting of water, buffer and enzyme. This was kept on ice until use.



The following program was ran in the PCR machine (program iGEM USER):

37 °C for 30 mins.

25 °C for 15 mins.

20 °C for 10 mins.

12 °C ∞

With no cycles.

2. GEL ELECTROPHORESIS

Team members: Claudia and Signe

The gel electrophoresis was run on the products of the USER ligation. The gel electrophoresis was according to the protocol from 23rd of May 2019.

Procedure

As we only had a small amount of sample we took 1.5 μL samples and loading dye μL.

Results

The gel was loading in the following order with the following samples:

Sample no. Nucleotide fragment
1 Ass2C + pTDH + TPSTI
2 Ass2C + pTDH + TPSTII
3 X3A + pCCW12 + HuLHCGR + li + sfGFP
4 X3A + pCCW12 + HuLHCGR
5 X3A + pCCW12 + GPER
6 X3A + pCCW12 + XLHCGR
7 X3A + pCCW12 + XLHCGR+ li+ sfGFP
8 Ass2A + pPGKI + Gαs

Conclusion

User ligation was seen on sample 1, 3, 4, 5, 6, 7 and 8 by a band around 7000 bp. User ligation needs to be done for sample 2 again.

3. YEAST STREAKING




Team members: Claudia and Signe

The following liquid colonies were streaked on plates:

Plate no. Colony no. Date
2 4 22/07
2 7 22/07
3 3 22/07
5 6 01/08

*Created date of the plate.

Procedure

  1. Four Glu-U agar plates were casted.
  2. The liquid colonies were streaked each on one plate.
  3. The plates were incubated O/N at 30 degrees.

4. GLYCEROL STOCKS




Team members: Claudia and Signe

Glycerol stocks were made for the following fragments:

Plate no. Colony no. Date
2 4 22/07
2 7 22/07
3 3 22/07
5 6 01/08

*Created date of the plate.

Materials

  • 50% glycerol
  • Overnight cultures
  • Cryotubes

Procedure

  1. Add 500 µL 50% glycerol to each of the cryotubes (one cryotube to one sample of fragment)
  2. Add 500 µL of each of the O/N cultures to the respective tubes.
  3. Put the cryotubes in the -80 degree Celsius freezer.

5. PREPARATION OF OLD SAMPLES FOR SEQUENCING




Team members: Swenja

The following samples have been prepared for sequencing:

Sample nr. Construct Date of creation
I - C1 Ass2A - pPGK1 - GPAIGαs 06/08
I - C3 Ass2A - pPGK1 - GPAIGαs 06/08
I - C5 Ass2A - pPGK1 - GPAIGαs 06/08
I - C7 Ass2A - pPGK1 - GPAIGαs 06/08
II - 1 X3A - pCCW12 - GPER 24/07
II - 2 X3A - pCCW12 - XLHCGR 24/07
II - 4 Ass2A - pPGK1 - GPAIGαi 24/07
II - 6 Ass2A - pPGK1 - GPAIGαs 24/07

Procedure

  1. The samples were mixed according to the table below. The amount of sample added (in µl) was calculated based on the DNA concentrations that have been measured with NanoDrop.
    Vector µl corresponding to 500 ng
    Primers 3.3 µl
    Water x µl
    Total 10 µl
  2. For each sample three tubes with different primers were prepared.
Sample no Construct 1. Primer 2. Primer 3. Primer
I Ass2A - pPGK1 - GPAIGαs YEA75 YEA137 GPAI-F
II - 1 X3A - pCCW12 - GPER YEA74/td> YEA81 GPER-F
II - 2/td> X3A - pCCW12 - XLHCGR YEA74 YEA81 XLHCGR-F
II - 4 Ass2A - pPGK1 - GPAIGαi YEA75 YEA137 GPAI-F
II - 6 Ass2A - pPGK1 - GPAIGαs YEA75 YEA137 GPAI-F

Results

The sequences can be found on the google drive with the sequencing nr. According to the table below.

Sample no Construct 1. Primer 2. Primer 3. Primer
I - C1 Ass2A - pPGK1 - GPAIGαs 1D89ZAF228 1D89ZAF229 1D89ZAF230
I - C3 Ass2A - pPGK1 - GPAIGαs 1D89ZAF231 1D89ZAF232 1D89ZAF233
I - C5/td> Ass2A - pPGK1 - GPAIGαs 1D89ZAF234 1D89ZAF235 1D89ZAF236
I - C7 Ass2A - pPGK1 - GPAIGαs 1D89ZAF237 1D89ZAF238 1D89ZAF239
II - 1 X3A - pCCW12 - GPER 1D89ZAF240 1D89ZAF241 1D89ZAF242
II - 2 X3A - pCCW12 - XLHCGR - GPAIGαi 1D89ZAF243 1D89ZAF244 1D89ZAF245
II - 4 Ass2A - pPGK1 - GPAIGαs 1D89ZAF246 1D89ZAF247 1D89ZAF248
II - 6 Ass2A - pPGK1 - GPAIGαs 1D89ZAF249 1D89ZAF250 1D89ZAF251

About Us

We are Ovulaid: a team of 13 students from the University of Copenhagen working on a novel ovulation detection system, using synthetic biology.

Keep in Touch

iGEM Team Copenhagen

iGEM_Copenhagen

iGEM_Copenhagen

UCPH.IGEM2019@gmail.com

Address

University of Copenhagen

Thorvaldsensvej 40, Frederiksberg C

Denmark