Team:UCopenhagen/Notebook/Week 35
Week 35 (26th of August-1st of September)
26th of August
PLASMID PURIFICATION
Team members:
We did miniprep on the following samples using the protocol from the miniprep kit. Samples:
- GPER 1.1
- GPER 2.1
- GPER-sfGFP 2.2
- GPER sfGFP 2.3
- Gαi
- STE12
- ZsGreen
- Gαi [3]
- STE12 [7]
- ZsGreen [9]
28th of August
1. Yeast colony PCR
Team members: Swenja and Noël
Procedure
Yeast colony PCR was performed on the following plates that were created on the 22nd of August
- pCCW12-XLHCGR-sfGFP+BASS2 + X3C
- pCCW12-XLHCGR-X3A+Ass2A-pPGK1-GPA1-Galphas+Ass2B-pRET2-STE12
- X3A-pCCW12-XLHCGR+Ass2A-pPGK1-GPA1-Galphai+Ass2B-pRET2-STE12
- X3A-GPER+pCCW12-Ass2A-pPGK1-GPA1-Galphas+Ass2B-pRET2-STE12
- X3A-GPER+pCCW12-Ass2A-pPGK1-GPA1-Galphai+Ass2B-pRET2-STE12
- X3A-pccW12-GPER-sfGFP+Ass2A
The colony PCR was conducted as described before (see for example 26th of July, with the difference that the elongation time was 2.5, instead of 1.5 minutes.) with 10 samples of each plate.
Note: samples 1.6 was confused with 1.9 and 2.2 with 2.4 while taking the samples from the plate.
The PCR was conducted, but the gels were not loaded yet due to time reasons. That was done the following day.
29th of August
1. Yeast colony PCR
Team members: Swenja and Anett
For the yeast transformation we prepared Glu-u agar (2x500ml) and we made 8 plates as well.
Material
For Glu-u (500 ml):
Set pH with 10 drops of 10% NaOH and then fill it up to 500 ml.
Procedure
- Mix everything in a 500ml bottle, set pH and fill it up to 500ml. Put it into the autoclave.
- After autoclaving Noël added Trp, His, Leu amino acids to the media (the amounts are for 2x 500ml media!): 40mg Leu, 40mg Trp, 20mg His were dissolved in 20ml MQ water. 10 ml was added to each bottle of media under the sterile bench using a syringe and a sterile filter. So each bottle had 10mg/L His, 20mg/L Trp and Leu.
- We made 8 plates from the media under a sterile bench and put the rest of the media into the cupboard.
2. Run the gel of Colony PCR Samples from 28/8
Team members: Swenja and Anett
Procedure
4ul of each sample were mixed with 1ul loading dye and loaded into the well of the gels.
Result
We got positive bands for:
2.2 and 2.4 from pCCW12-XLHCGR-pPGKI-GPAIGalphas-pRET2-STE12-pFIG1-ZsGreen
1.1, 1.2, 1.3 from pCCW12-XLHCGR-sfGFP
4 from pCCW12-GPER-pPGK1_GPAI-Galphas-pRET2-STE12-pFIG-ZsGreen
3.Plasmid ligation
For the yeast transformation we made plasmid ligation with the different fragments. (For sample 3 we used a wrong sample so we did not use it further on for the yeast transformation)
Material
| Sample | Concentration [ng/µl] |
|---|---|
| pCCw12-XLHCGR-X3A | 468.9 |
| X3A-pCCW12-GPER | 367.8 |
| pPGK1-GPAI-Galphai-Ass2A | 404.0 |
| pRET2-STE12-Ass2B | 281.3 |
| pFIG1-ZsGreen-X3C | 220.5 |
| BAss2 | 287.5 |
| X3C | 286.8 |
| Ass2C | 176.6 |
| X3A-pCCW12-GPER-sfGFP | 157.9 |
For the 5 assemble system we need to calculate ul corresponding to 1000ng for the receptors and 1500ng for all the other backbones. For the 3-asseble system we need to calculate ul corresponding to 600ng for the receptors and 900ng for all the other backbones.
Procedure
| System nr. | X3A modula | µL | BAss2 module | µL | Ass2B module | µL | Ass2C module | µL | X3C module | µL | Total |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 3 | pCCW12-XLHCGR | 2.1 | pPGK1-GPAI_Galphai | 3.7 | pRET2-STE12 | 5.3 | Ass2 C | 5.6 | pFIG1-ZsGreen | 6.8 | 23.5 |
| 5 | pCCW12-GPER | 2.7 | pPGK1-GPAI-Galphai/td> | 3.7 | pRET2-STE12 | 5.3 | Ass2 C | 5.6 | pFIG1-ZsGreen | 6.8 | 24.1 |
| 6 | pCCW12-GPER-sfGFP | 3.8 | Bass 2A/td> | 3.1 | - | - | - | - | X3C | 3.1 | 10 |
The samples were mixed according to the table. 1/9 of Cut Smart buffer (of total amount) was added to each sample and 1ul of Not1 enzyme.
| System nr.. | Buffer added (µL) |
|---|---|
| 3 | 2.6 |
| 5 | 2.7 |
| 6 | 1.1 |
We started the following program in the PCR machine:
- 37 °C for 2 hours
- 65 °C for 20 min.
- 12 °C indefinitely
The samples where stored in -20 °C fridge.
4. Incubation of positive yeast colonies
The positive yeast colonies from 29/8 were inocculated in 5ml Glu-u media at 30°C in the shaking incubator.
Material
2.2 and 2.4 from pCCW12-XLHCGR-pPGKI-GPAIGalphas-pRET2-STE12-pFIG1-ZsGreen
1.1, 1.2, 1.3 from pCCW12-XLHCGR-sfGFP
4 from pCCW12-GPER-pPGK1_GPAI-Galphas-pRET2-STE12-pFIG-ZsGreen.
5. Inoculation of yeast culture
We inoculated the yeast cultures for the yeast transformation tomorrow.
Procedure
One colony of seed culture was resuspended in 5ml YPD media and incubated at 30°C in the shaking incubator ON for further use in the yeast transformation.
30th of August
1. Yeast transformation
Team members: Swenja and Anett
For the yeast transformation we used sample 5 and 6 from 29th of August.
Material
YPD media
Yeast liquid culture
0.1 LiOAc
Salmon sperm (ssDNA)
PLI (1 part 1M LiOAc and 9 part 50% PEG)
Sterile water
Glu-u plates
Prodecure
- 0.9ml YPD media and 0.1ml of ON yeast culture were mixed in a cuvette under sterile bench. OD600 was measured, YPD was used as a blank. OD600=0.178, OD of the original sample: 1.78
- We diluted the culture to an OD600=0.25. c1*V1=c2*V2 which means 25*0.25/1.78=3.51ml. In a falcon tube we added 21.5ml YPD and 3.51ml of the ON culture were mixed to get a total of 25ml culture with an OD600=0.25.
- Culture was incubated for another 4 h. We checked the OD with the same method how we did it in the 1.) step. The OD600 for the culture was unfortunately 1.48 which is a bit too high.
- The ssDNA (salmon sperm) was boiled for 5 min at 99°C in the heating block and later kept on ice.
- The culture was centrifuged in a 50ml falcon tube at 3000rcf for 10min at room temperature (RT).
- The supernatant (SN) was poured into a waste tube with one movement and the pellet was resuspended in 1ml of 0.1 LiOAc. The whole soultion was transferred into a 1.5ml eppendorf tube.
- The culture was centrifuged at 7000rcf for 1 minute at RT and the SN was removed with a pipette.
- The pellet was resuspended in 100ul of 0.1M LiOAc and 30ul of salmon sperm was added and mixed carefully.
- 1ml of PLI was added and mixed well.
- The DNA samples from the ligation from 29/8 (5th and 6th sample) were transferred into an eppendorf tube and to each sample 200ul of the yeast cells was added. Mix it well.
- The samples were heat shocked for 30 minutes at 42°C in the heating block at 750rpm.
- The cells were spin down at 7000rcf for 1min at RT and the SN was descarded.
- The pellet was resuspended in 1ml sterile water and mixed well.
- Once more the cells were centrifuged. This time: 8000rcf for 1min, RT.
- 900ul of the SN was removed and the pelled was resuspended in the rest of the water.
- Transformed cells were plated on Glu-u plates and the plates were kept in the incubator for 3 nights at 30 °C on the second floor.
Results
We checked the plates on the 2sn of September. Unfortunately, we had too many colonies on both of them so we could not use it for colony PCR (no separate colonies). Since they looked weird, we throw them out.
2. Glycerol stocks
Team members: Noël and Signe
We did glycerol stocks of the positive samples from the colony PCR from the 28th of August (gel on the 29th). We used the liquid cultures of them made on the 29th of August.
Material
29/8 S4C3 Yeast GPER-Galphas-STE12-ZsGreen
29/8 Positive colony 2.3 Yeast XLHCGR-Galphas-STE12-ZsGreen
29/8 S1C2 Yeast XLHCGR-sfGFP
29/8 S1C1 XLHCGR-sfGFP
29/8 S2C4 XLHCGR-Galphas-STE12-ZsGreen
29/8 S1C3 Yeast XLHCGR-sfGFP
Procedure
The stocks were made by taking 600ul 50% glycerol i H2O and 600ul sample. For the sample marked with a star too much glycerol was taken and therefore more sample was taken as well. Here was taken about 800ul glycerol and 900ul sample.
The samples were taken to the -80°C freezer.
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
