Team:UCopenhagen/Notebook/killswitch/week39

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Kill Switch Week 39 (23rd-29th of September)

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25th of September

Galactose induction assay of pGAL1-BAX (liquid)

Team members: Hitesh & Swenja

A galactose induction assay was performed in 96-well plates for the yeast containing the pGAL1-BAX construct. A control was prepared with a yeast strain containing the empty vector.

Materials

• 96-well plates
• O/N pGAL1-BAX culture
• O/N control culture (Yeast with empty vector)
• Raff media w/ 1% gal
• Raff media w/o gal
• Sterile galactose stock (20%; 20 g/l)

Procedure

1. A 20% galactose stock was made by dissolving 0.2 g galactose in 1 ml raff media and filter sterilising it through a 0.22 µm filter.
2. A 2% galactose stock was then made from the 20% stock by mixing 100 µl of the stock with 900 µl raff media w/o gal.
3. The wells of two 96-well plates were filled with galactose stock (20% for all concentrations but 0.05% gal and 0.01% gal. Here, the 2% gal stock was used) and raff media according to the table below. For both pGAL1-BAX and the control duplicates were made and the wells filled in such a way, that the galactose concentration increased from left to right.
4. 100 µl of the O/N cultures were mixed with 900 µl raff media w/o gal in cuvettes, respectively. The OD at 600 nm was measured and the cultures diluted to an OD of 0.5.
5. 110 µl of cells were added to each well. Besides, two wells of each plate were filled with 140 µl raff media w/o galactose.
6. The plates were incubated O/N in the 30 °C shaking incubator before the OD was measured in the plate reader.

Gal concentration [%]	V (Gal stock, 20%) [µl]	V (Raff media) [µl]	V (cells) [µl]	V (Total) [µl]
4	28	2	110	140
3	21	9	110	140
2	14	16	110	140
1	7	23	110	140
0.5	3.5	27	110	140
0.2	1.4	29	110	140
0.1	0.7	29	110	140
0.05	3.5*	27	110	140
0.01	0.7*	29	110	140

*2% galactose stock used

Results

Unfortunately, the results were very inconsistent. Therefore, we decided to repeat the assay in 24-well plates with a higher volume to minimise pipetting errors.

27th of September

Inoculation of E.coli containing pTEF-BI-1 and pGal1-BI-1

Team members: Noel & Jonas

Materials and Procedure

The cultures that were picked earlier the same day by Ben for Colony PCR were incoulated at 30° C in LB+Amp liquid media.

Galactose induction assay (liquid)

Team members: Anett & Swenja

As the galactose induction assay performed in a 96-well plate hasn’t shown precise enough results, we have decided to perform another assay with 24-well plates as these allow for higher volumes and with that higher accuracy. The assay was to be performed with both the pGAL1-BAX and the pTEF1-BAX-pGAL1-BI-1 construct with a yeast culture containing the empty vector as a control. Unfortunately, the culture with the pGAL1-BAX construct didn’t grow sufficiently.

Materials

• 24-well plates
• O/N pTEF1-BAX-pGAL1-BI-1 culture
• O/N control culture (Yeast with empty vector)
• Raff media w/ 1% gal
• Raff media w/o gal
• Sterile galactose stock (20%; 2g/ml)

Procedure

1. 100 µl of the O/N cultures were mixed with 900 µl Raff media w/ 1% gal in cuvettes, respectively. The OD at 600 nm was measured and the cultures diluted to an OD of 0.5 and kept on ice. The control was diluted in raff media w/o galactose. The pTEF1-BAX-pGAL1-BI-1 culture was diluted in raff media w/ 1% gal.
2. A 2% galactose stock was made from the 20% stock by mixing 100 µl of the stock with 900 µl raff media w/o gal.
3. The wells were filled with galactose stock (20% for all concentrations but 0.05% gal and 0.01% gal.Here, the 2% gal stock was used) and raff media according to the table below. For both the killswitch and the control duplicates were made and the wells filled in such a way, that the galactose concentration increased from left to right.
4. The killswitch culture was washed shortly before use by centrifuging the cells down (highest speed on falkon tube centrifuge for appr. 4 mins.) and resuspending them in raff media w/o galactose.
5. 786 µl of cells were added to each well. Besides, two wells of each plate were filled with 1 ml raff media w/o galactose.
6. The OD at 600 nm was measured in the plate reader. The wells with media were used as blank. (If yeast sinks down and form small white spots at the bottom, shake a bit before measuring OD)
7. The plates were then incubated O/N in the 30 °C shaking incubator before the OD was measured again and compared to T0.

Gal concentration [%]	V (Gal stock) [µl]	V (Raff media) [µl]	V (cells) [µl]	V (Total) [µl]
4	200	14	786	1000
3	150	64	786	1000
2	100	114	786	1000
1	50	164	786	1000
0.5	25	189	786	1000
0.2	10	204	786	1000
0.1	5	209	786	1000
0.05	25	189	786	1000
0.01	10	209	786	1000

Results

Unfortunately, the end ODs were very random why we decided to repeat the assay.

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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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