Team:UCopenhagen/Notebook/killswitch/week37

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Kill Switch Week 37 (9th-15th of September)

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9th of September 2019

1. Second Gal-induction assay

Team members: Noël & Jonas

Purpose of this experiment is to see if the GAL1-BAX cells can be killed by exposing them concentrations of Galactose.

Materials

• A cell culture with cells expressing pGal1-Bax that was cultivated in Raffinose media.
• Plates that contain Raffinose as energy source and different amounts of Galactose as inducer
• The following concentrations were measured: 0, 0.1, 0.5, 1, 2 g/l

Procedure

1. The culture was diluted to an OD of 0.5.
2. A 100x and a 1000x dilution of this stock solution were created.
3. The 50 µl of these dilutions were put onto the aforementioned plates. Duplets for each dilution-Gal-concentration combination were created.
4. The plates were incubated at 30° C for three nights.

Results

Unfortunately, this experiments again resulted in high colony numbers on the plates. While the plates were still in the incubator, better experiments were chosen to show the galactose-induced dead of the cell. This experiment has not been repeated afterwards, as systematic flaws and problems with the culture were discovered.

10th of September 2019

1. Plasmid digestion

Team members: Swenja

The parts for the different assembler systems were mixed and the plasmids digested for use in the yeast transformation the next day.

Materials

• Gene constructs
• XI2C backbone
• CutSmart buffer
• Enzyme Not1

Procedure

1. The needed amounts in µl of each construct had to be calculated. For the 3-assembler system we want 600 ng of the construct in the first backbone (XI2A) and 900 ng for the two other constructs/empty vectors to be added.
2. The constructs/empty vectors were then mixed together in a PCR tube according to the table below.
3. The total volume of each tube was calculated and the amount of CutSmart buffer responding to 1/9 of this volume added.
4. Lastly, 1 µl of Not1 enzyme was added to each tube and the content mixed and briefly span down.
5. The samples were then placed in a PCR machine with the following settings (YTdigest):
   1. 37 °C for 2 h
   2. 65 °C for 20 mins
   3. 12 °C indefinitely

Assembler system nr.	XI2A module	µl	B module	µl	XI2C module	µl	Total volume (µl)
1.	pTEF1-BAX	7.7	pGAL1-BI-1	10.6	XI2C	18.9	37.2
2.	pADH2-BAX	9.5	pGAL1-BI-1	10.6	XI2C	18.91	39

Sample nr.	Total volume (µl)	Buffer added (µl)	Not1 added (µl)
1.	37.6	4.1	1
2.	39.4	4.3	1

12th of September 2019

1. Sample digestion for yeast transformation

Team members: Swenja

Sample digestion was repeated

Materials

• Gene constructs
• XI2C backbone
• CutSmart buffer
• Enzyme Not1

Procedure

1. The needed amounts in µl of each construct had to be calculated. For the 3-assembler system we want 600 ng of the construct in the first backbone (XI2A) and 900 ng for the two other constructs/empty vectors to be added.
2. The constructs/empty vectors were then mixed together in a PCR tube according to the table below.
3. The total volume of each tube was calculated and the amount of CutSmart buffer responding to 1/9 of this volume added.
4. Lastly, 1 µl of Not1 enzyme was added to each tube and the content mixed and briefly span down.
5. The samples were then placed in a PCR machine with the following settings (YTdigest):
   1. 37 °C for 2 h
   2. 65 °C for 20 mins
   3. 12 °C indefinitely

Assembler system nr.	XI2A module	µl	B module	µl	XI2C module	µl	Total volume (µl)
1.	pTEF1-BAX	7.7	pGAL1-BI-1	10.6	XI2C	18.9	37.2
2.	pADH2-BAX	9.5	pGAL1-BI-1	10.6	XI2C	18.91	39

Sample nr.	Total volume (µl)	Buffer added (µl)	Not1 added (µl)
1.	37.6	4.1	1
2.	39.4	4.3	1

2. Setting up the fresh yeast for transformation

Team members: Noël

Untransformed yeast was put into 5 ml of fresh YPD media and left over night at 30° C.

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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.

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