Team:UCopenhagen/Notebook/killswitch/week36

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Kill Switch Week 36 (2nd-8th of September)

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5th September 2019

1. Set up a culture of the XI2C vector

Team members: Noël & Anett

Materials

• Karel gave us the E.coli with XI2C from his glycerol stock.

Procedure

1. Under the steril bench we poured into a sterile falcon tube 5 ml LB media+Car and with a pipette tip we added a little amount from the E.coli glycerol stock. We put it into the incubator for 37°C ON.

2. Gal-induction assay: Media preparation

Team members: Noël & Anett

Materials & procedure

We prepared the following cultures:

• Rafinose basic media 1 (=RBM1): 20g rafinose, 6.7g yeast nitrogen base, 1.34g synthetic drop out media, 0.04g Trp, 0.04g Leu, 0.02 His, fill it up to 1l and set pH with 20 drops of 10% NaOH
• Rafinose basic media 2 (=RBM2): Exactly the same, just half the amounts were used of each component
• Raffinose-Full-GAL (10 g/l) (=RBMFG): 100ml RBM1 + 1g GAL
• RBM+10 g/l GAL+Agar: 100ml RBM1 + 1g GAL + 2g Bak. Agar
• RBM+1 g/l GAL+Agar: 90ml RBM + 10ml RBMFG+ 3g Bak. Agar
• RBM + 0.5 g/l GAL + Agar: 95ml RBM + 5ml RBMFG+ 3g Bak. Agar
• RBM + 0.1 g/l GAL + Agar: 99ml RBM + 1ml RBMFG+ 3g Bak. Agar
• RBM + 0.01 g/l GAL + Agar: 99.9 ml RBM + 0.1 ml RBMFG+ 3g Bak. Agar
• RBM + Agar: 100 ml RBM1 + 2g Bak. Agar

6th September 2019

1. Gal-induction assay: Preparing plates

Team members: Jonas & Noël

Materials & procedure

• The all the agar-containing media that were prepared the day before. Four plates were done with each bottle.

2. Gal-induction assay: Prepare a culture and plate it

Team members: Jonas & Noël

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, 0.01, 0.1, 0.5, 1, 10 g/l

Procedure

1. The culture was diluted to an OD of 0.5.
2. 50 and 150 µl of this culture are plated. Duplets for each Volume-Gal-concentration combination were created.
3. The plates were incubated at 30° C for three nights.

Results

Unfortunately, the plates were overgrown, because too much cells were applied on the plates. But the general trend was obvious. The plates with the higher sugar concentrations seem to show less growth. However, this is not definitely quantifiable. It was decided to repeat the experiments.

3. Purification of XI2C

Team members: Signe

Materials & procedure:

• The plasmid was harvested from the culture that was set up the afternoon before using the EZNA plasmid purification kit. The purified plasmid was measured with the NanoDrop. The final concentration was 47.5 ng/µl

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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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