Team:UCopenhagen/Notebook/killswitch/week41





Kill Switch Week 41 (7th-13th of October)

7th of October

Inoculation for gal-induction assay

Team members: Swenja

A fresh culture of yeast with pGAL1-BAX and the empty vector, respectively, were prepared for the next day.

Materials and Procedure

0.5 ml of a former culture of yeast with pGAL1-BAX was added to 4.5 ml fresh Raff-U media w/o gal. The culture was put into the 30 °C shaking incubator O/N. Same was done for a control culture containing the empty vector. Additionally, 1 ml of the empty vector culture was added to 9 ml Glu-U media, incubated O/N at 30 °C in the shaking incubator and stored in the fridge the next day.

8th of October

Sequencing

Team members: Jonas

Sequencing was done on ADH2-BAX colony 3.1, GAL1-BAX colony 1.2, GAL1-BI-1 colony 4 and TEF1-BI-1 colony 2

Procedure

  1. 500 ng of DNA was used or 6,7 microliter if the concentration was to low to get 500 ng in less than 6,7 microliter. 3,3 microliter of primer was used in each sample. The samples were filled to 10 microliters with water.
  2. Each construct was sequenced from both the 5' end and the 3' end because the constructs are too large for one sequencing run to cover them. One primer was used for each sample which means we had two samples for each construct.

Results

The GAL-BAX sample was perfect with no problems. The GAL-BI construct had some sequence errors at the ends of the sequencing data which is likely a problem with the sequencing technology.

Gal-induction assay (liquid) of pGAL1-BAX

Team members: Swenja

Due to inconsistent ODs during the last assay, the liquid gal-induction of pGAL1-BAX was repeated.

Materials

  • 24-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 (40%; 400 g/l)

Procedure

  1. A 20% galactose stock was made using the 40 % galactose stock that recently has been prepared. For this, 2,5 ml of the 40% galactose stock were mixed with 2,5 ml raff media w/o galactose.
  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 24-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. 786 µl of cells were added to each well and individually mixed. Besides, two wells of each plate were filled with 1 ml raff media w/o galactose.
  6. Immediately, the OD at 600 nm was measured using a plate reader. The wells with media were used as blank. Before the reading the plates were gently mixed by up and down movements on the table.
  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

Results

Unfortunately, the start OD of 0.5 was too high. Therefore, the difference between the concentrations were insignificant.

Preparing plates for gal-induction assay of the plasmid constructs

Team members: Noel and Swenja

Plates have been prepared for the upcoming galactose-induction assay of the killswitch constructs on plasmids. All plates were pipetted using the 40% galactose stock and Raff-U-W media w/o galactose. The total volume of each plate was 15 ml. The following plates have been prepared: 14 plates of Raff-U-W w/ 1% galactose, 14 plates of Raff-U-W w/ 0.5% galactose, 14 plates of Raff-U-W w/ 0.1% galactose.

10th of October

Dual plasmid transformation

Team members: Jonas and Hitesh

We performed multiple different transformation of yeast with each yeast being transformed with two plasmids (see table below).

Yeast nr. Plasmids (+selection marker)
1 Empty vector (EV)(+u) + EV (+w)
2 TEF1-BAX + GAL1-BI-1
3 ADH2-BAX + GAL1-BI-1
4 EV (+u) + GAL1-BI-1
5 TEF1-BAX + EV (+w)
6 ADH2-BAX + EV (+w)
7 EV (+u) + TEF1-BI-1
8 TEF1-BAX + TEF1-BI-1
9 ADH2-BAX + TEF1-BI-1
10 GAL1-BAX + EV (+w)

The plasmids containing BAX all carried a uracil selection marker and the plasmids containing BI-1 all had a tryptophan selection marker

Materials

  • Yeast O/N culture
  • Purified plasmids
  • ssDNA (salmon sperm)
  • 0.1 M LiAc
  • 50 % PEG
  • PLi (1 part 1 M LiAc and 9 parts of 50 % PEG)
  • Heat block
  • Sterile bench
  • Pipette
  • Pipette tips
  • Eppendorf tubes
  • Growth plates
  • Centrifuge for falcon tubes
  • Centrifuge for eppendorf tubes
  • 30 degrees incubator

Procedure

  1. The ssDNA is boiled for 5 min at 99 degrees and then but directly on ice.
  2. The o/n culture is diluted so it has an OD600 of 0.25 and then it is put into the incubator for 4 hours so it reaches an OD of roughly 1. 25 ml of culture is good for 4-5 transformations.
  3. The culture is then spun down in the centrifuge at 3000 rcf for 10 min.
  4. The supernatant is discarded
  5. The pellet is resuspended in 1 ml of 0.1 M LiAc and the solution is then transferred to an eppendorf tube
  6. The eppendorf tube was spun at 7000 rcf and the supernatant was removed with a pipette.
  7. The pellet is resuspended in 100 microliter of 0.1 M LiAc and mixed with 30 microliter of ssDNA.
  8. 1 ml of PLi is added to the samples
  9. 200 microliter of cells were added to the DNA which the cells are to be transformed with. We used 300 ng of plasmid
  10. The cells were then heat shocked at 42 degrees for 30 min.
  11. The cells were then spun at 7000 rcf for 1 min and the supernatant was removed.
  12. The cells are resuspended in 1 ml of water and spun at 8000 rcf.
  13. 900 microliter of water is removed and the cells are then resuspended in the remaining 100 microliter and then plated on selection plates (lacking the markers present on the plasmids) with or without galactose.

Results

Yeast nr. Plasmids (+selection marker) Result
1 Empty vector (EV)(+u) + EV (+w) Growth on both plates
2 TEF1-BAX + GAL1-BI-1 No growth on either plates with or without galactose
3 ADH2-BAX + GAL1-BI-1 We saw growth but the colonies were very small. The plate with galactose had a lot more growth than the plate without galactose
4 EV (+u) + GAL1-BI-1 We only saw a few colonies on the plate with galactose and we saw good growth on the plate without galactose
5 TEF1-BAX + EV (+w) We saw only a few colonies on both plates
6 ADH2-BAX + EV (+w) On the plate with galactose there was a few larger colonies and on the plate without galactose the colonies were smaller
7 EV (+u) + TEF1-BI-1 no colonies on the plate with galactose and a few colonies on the plate without galactose
8 TEF1-BAX + TEF1-BI-1 No colonies on either plate
9 ADH2-BAX + TEF1-BI-1 We saw a lot of growth on the plate with galactose but no growth on the plate without
10 GAL1-BAX + EV (+w) we saw growth on the plate without galactose but only a couple of colonies on the plate with galactose

11th of October

Preparation of plates for the plasmid construct assays

Team members: Swenja and Signe

Plates have been prepared for the upcoming assays of the killswitch constructs on plasmids. The plates with distinct galactose concentrations (apart from GOLD-1 plates) have been pipetted using 40% galactose stock, Raff-U(-W) media w/o galactose and Raff-U(-W) media w/ 1% galactose. The total volume of theses plates was 25 ml. The following plates were made:

  • BRONZE: 6x Raff-U w/ 0% galactose, 6x Raff-U w/ 1% galactose, 4x Raff-U w/ 0.05% galactose, 4x Raff-U w/ 0.1% galactose and 4x Raff-U w/ 0.3% galactose.
  • GOLD-1: 17x Raff-U-W w/ 0% galactose and 9x Raff-U-W w/ 1 % galactose
  • GOLD-2: 20x Glu-U-W
  • GOLD-3: 6x Raff-U-W w/ 1% galactose, 1x Raff-U-W w/ 0% galactose, 6x Raff-U-W w/ 0.5% galactose and 6x Raff-U-W w/ 0.1% galactose

Miniprep

Team members: Jonas

Purified plasmids containing ADH2-BAX, TEF1-BAX, GAL1-BAX, TEF1-BI-1 and GAL1-BI-1 from E. coli

Materials

  • Miniprep kit
  • Centrifuge
  • Eppendorf tubes
  • Nanodrop machine (to check concentration of DNA)

Procedure

Followed the protocol from the kit with the exception that I used twice as much o/n culture and twice as much of the reagents for the first three steps that required reagents from the kit.

Results

Sample Concentration (ng/µl)
1 154,4
2 184,1
3 179,3
4 182
5 83 (accidentally used twice as much elution buffer for this sample)
6 142,2
7 145,9
8 144,9
9 193,1
10 195,3
11 182,5
12 184,3

12th of October

Gal-induction assay of pGAL1-BAX

Team members: Swenja and Ojas

A quantitative gal-induction assay of pGAL1-BAX was made on plates.

Materials

  • O/N culture of yeast containing pGAL1-BAX
  • O/N culture of yeast containing in empty vector
  • Raff-U plates with 0%, 0.05%, 0.1% and 1% galactose
  • Raff-U media w/o galactose

Procedure

  1. The OD600 of both cultures was measured by mixing 100 µl of culture and 900 µl of Raff-U media in a cuvette.
  2. The OD600 was then adjusted to 0.5 in a total volume of 5 ml.
  3. A dilution series was made until a dilution of 10-4 by mixing 100 µl of culture with 900 µl Raff-U media w/o galactose.
  4. 100 µl of both the 10-3 and 10-4 dilution culture was spread on plates with 0%, 0.05%, 0.1% and 1% galactose, respectively. (Duplicates were made)
  5. The control culture was only spread on plates containing 1% galactose (10-3 and 10-4 dilution).
  6. The plates were incubated for 3 days at 30 °C.

Results

After incubation, the colonies on each plate were quantified and the CFU/µl were calculated.

Dilution 1% Galactose 0.3% Galactose 0.1% Galactose 0.05% Galactose Negative Control
Colony forming units (CFU)
10-3 1 13 13 12 20 Contaminated 21 26 300
10-4 - - - 4 4 6 2 4 38 50
CFU/ml 1.3 x 105 1.25 x 105 2.5 x 105 2.35 x 105 4.65 x 105

Inoculation of yeast O/N cultures for WB

Team members: The mystic lab elf

Following positive yeast O/N cultures were set up

  • GPER-sfGFP (plate 2, colony 4, and P2C7) (8/8)
  • XLHCGR-sfGFP (P3C3 and P5C6) (8/8)
  • Negative control (Empty vectors) from Nat
  • Positive control (Constitutive sfGFP) from Nat

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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iGEM Team Copenhagen

iGEM_Copenhagen

iGEM_Copenhagen

UCPH.IGEM2019@gmail.com

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