Team:UCopenhagen/Notebook/killswitch/week42

• 
• Project
  • Description
  • Design
  • Results
  • Future Plans
• Lab
  • Parts Overview
  • Protocols
  • Safety
  • Notebook
  • Modeling
• Human Practices
  • Integrated Human Practices
  • Entrepreneurship
  • App
  • Education and Engagement
• Collaboration
  • Collaborations
  • Meetups
  • Nordic iGEM Conference
• Team
  • Team Members
  • Team Building
• Acknowledgements
  • Attributions
  • Sponsors
• 
  • Judging form
  • Medal Requirements
  • Awards

Kill Switch Week 42 (14th-20th of October)

• Previous
• Index
• Next

14th of October

Restreak ADH2-BAX+GAL1-BI plasmid transformants

Team members: Jonas

Colonies from the ADH2-BAX+GAL1-BI plate were restreaked on both plates containing galactose and on plates without galactose.

Procedure

Some of a colony was picked with an inoculation loop and streaked on a plate. This was done for both plates containing galactose and on plates without galactose. 10 different colonies were streaked on the new plates. The plates were put into a 30-degree incubator

15th of October

Yeast transformation

Team members: Swenja and Jonas

Our stock yeast was transformed with the 3-assembly system ADH2-BAX construct and a plasmid containing GAL1-BI-1.

• pADH2-BAX (integrated) + pGAL1-BI-1 (in PWUS)
• XI2A + B(Ass2) + XI2C + PWUS

Yeast with genome integrated GAL1-BAX was transformed with a plasmid containing GAL1-BI-1.

• pGAL1-BI-1
• PWUS

Materials

• O/N culture
• Glu-U +Agar media
• Cuvettes and spectrophotometer for OD value check
• Falcon tubes
• Plasmid samples for all modules
• NotI enzyme
• CutSmart Buffer

Procedure

1. The O/N yeast culture grown in liquid YPD media was diluted 1:10 (100 µl culture + 900 µl YPD media only)
2. OD of the dilution was measured at 600 nm with pure YPD as standard blank
3. The undiluted O/N culture was then diluted to an OD of 0,25 for a total volume of 25 mL based on calculation on the OD of the diluted ON culture.
4. The 25 ml ON dilution (OD 0.25) was then kept in an incubator at 30 °C and 150 RPM for approximately 4 hours.
5. During the 4 hours digestion on the ADH2-BAX vector was performed. The samples were mixed according to the table below. As we have used the 3-assembly system, a volume corresponding to 600 ng DNA was used for the first backbone and a volume corresponding to 900 ng DNA of the other backbones. 1000 ng of the plasmids was used.
6. The DNA samples for integration were digested by NotI and left in the buffer for ligation. The enzyme was added as the last component.
7. The digestion samples were put in the PCR and the following program was run: 37 °C for 1 h 30 min, 65 °C for 20 min and 12 °C indefinitely
8. After inactivation of Not1, a volume corresponding to 1000 ng DNA of the plasmid was added to the samples for the first transformation. 100 ng of the plasmids for the transformation with the already integrated pGAL1-BAX were added directly.
9. Salmon sperm DNA was denatured by putting it on a heat block at 99 °C
10. When the desired OD was reached, the cultures ∆5 were transferred from the erlenmeyer flasks to Falcon tubes.
11. These were centrifuged at 3000 rpm for 10 min
12. 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.
13. The supernatant from the centrifuged Falcon Tubes containing was discarded (this was just poured out)
14. The pellet was resuspended in 1 mL Lithium acetate (with water)
15. The resuspension (1 mL) was transferred into an eppendorf tube
16. These were centrifuged at 7000 rpm for 1 min and the supernatant was discarded (by pipetting).
17. The pellet was resuspended in 100 µL Lithium acetate (with water) (Tip: resuspend with big pipette to stress the cells less)
18. 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.
19. The PLI was mixed by being inverted (not shaken because the PLI is hydroscopic)
20. 1 mL of PLI was put into the eppendorf tubes (PLI stabilizes the cells). It was homogenized by pipetting up and down.
21. The digested and ligated plasmids were transferred to eppendorf tubes.
22. 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.
23. The samples were heat shocked at 42 °C for 30 min at 750 rpm
24. After being heat shocked, the samples were centrifuged for 1 min at 7000 rpm and the supernatant was discarded.
25. 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)
26. They were again centrifuged for 1 min at 8000 rpm and 900 µL of the supernatant was discarded
27. The cells were resuspended in the remaining 100 µL and they were mixed gently
28. They were plated (and named according to the DNA samples) and kept in an incubator so colonies could grow.

17th of October

Restreaks from Monday were checked

Team members: Jonas

The restreaks from Monday were checked and only one set of plates (with and without galactose) showed the expected result (yeast growing on galactose plates and dying without galactose) and new restreaks were made on the 18th of October to confirm.

Gal-induction spot test of integrated and plasmid-expressed pGAL1-BAX

Team members: Swenja

To compare the effect of genome integrated and plasmid-expressed pGAL1-BAX, we have performed a spot test that uses different dilutions of the cultures as well as different concentrations of galactose on the plates.

Materials

• O/N yeast culture of genome integrated pGAL1-BAX
• O/N yeast culture containing the empty vector XI2A
• O/N yeast culture of pGAL1-BAX in PUUS
• O/N yeast culture containing the empty plasmids PUUS and PWUS
• Raff-U-W media
• Raff-U-W plates with 0%, 0.025%, 0.05%, 0.1% and 0.2% galactose

Procedure

1. The OD600nm of the O/N cultures was measured by diluting 100 µl culture in 900 µl raff-U-W media in a cuvette.
2. The O/N cultures were then diluted to an OD600nm of 0.5 in a total volume of 5 ml.
3. Dilution series were prepared in raff-U-W media for all cultures up to a dilution of 10-4.
4. On plates with 0%, 0.025%, 0.05%, 0.1% and 0.2% galactose, 10 µl of the 10-1, 10-2, 10-3 and 10-4 dilutions were spotted next to each other. The pGAL1-BAX cultures and their respective control culture were spotted on the same plate. Duplicates were made.
5. The plates were left to dry under the sterile bench and subsequently incubated for three days at 30 °C.

Results

18th of October

Restreak of restreak was made

Team members: Jonas

One of the restreaks which was checked yesterday was streaked onto new plates with and without galactose.

Materials

• Plate without galactose (Glu - U)
• Plate with galactose (RAF + 1 % galactose)

Procedure

1. One colony was restreaked from one of the restreaked plates which was made on the 14th of October. The plate had also been checked on the 17th.

Results

The colony that was restreaked on two plates grew on both plates with and without galactose. The may have inhibited the BAX gene in some way

BAX rescue assay

Team members: The dope ass bozos that are still in the lab

To see whether BI-1 can rescue the effect we have conducted a BAX rescue assay.

Materials

• Transformation plate of pGAL1-BAX (GI) + pGAL1-BI-1 (pWUS)
• Transformation plate of pGAL1-BAX + pWUS
• Sterile water

Procedure

1. Three colonies of each transformation plate were picked dissolved in 1 ml sterile water.
2. The OD at 600 nm was measured by transferring 0.5 ml of each culture into cuvettes. The ODs were all around 0.035 and considered equalised.
3. Dilution series were made for all cultures until a dilution of 10-3.
4. On both glu-U-W and raff-U-W (1% galactose) plates, 10 µl of the undiluted cultures and the three dilutions were spotted next to each other. Each culture was spotted along with one of the control cultures.
5. The plates were then incubated at 30 °C for three days.

Results

As expected, both cultures grew normally on the glu-U-W plates. However, against our expectations, there was strongly inhibited growth on the raff-U-W plates containing galactose. As the control strain only has BAX under the pGAL1 promoter, this is consistent with our predictions. In the other strain though, galactose induced expression of BI-1 should have rescued cell apoptosis. These results suggest that the colonies that have been picked for this assay were either not transformed with BI-1 (and only kept the markers) or BI-1 is not able to prevent BAX induced apoptosis in our yeast strain and is therefore not suitable for our kill switch.

• Previous
• Index
• Next

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