Team:UCopenhagen/Notebook/killswitch/week40





Kill Switch Week 40 (30th of September-6th of October)

2nd of October

USER ligation

Team members: Hitesh and Anett

USER ligation was performed to create the following constructs/samples:

  1. GAL1-BI1 in W plasmid
  2. TEF1-BI1 in W plasmid
The plasmids containing the promoters had been previously digested by Jon.

Materials

  • 10X USER enzyme
  • 10X CutSmart Buffer
  • Promoter fragments
  • Gene fragments
  • Linearised vector backbones

Procedure

  1. A MasterMix was created including water, CutSmart buffer and the USER enzyme according to the table below.
  2. For each ligation, the gene and promoter fragments were mixed in a PCR tube.
  3. 7 µl of MasterMix were added to each tube. Subsequently, the samples were mixed, span down and kept on ice.
  4. Finally, the samples were placed into a PCR machine with the following settings:
    1. 37 °C for 30 min
    2. 25 °C for 15 min
    3. 20 °C for 10 min
    4. 12 °C indefinitely
Reagent Amount (µl)
USER enzyme 1
CutSmart buffer 1
Promoter 1
Gene 1
Vector backbone 1
Water 5
Total 10

Inoculation of cultures for galactose induction assays

Team members: Swenja

1 ml of the cultures inoculated on the 1st October 2019 (yeast with empty vector and yeast with pGAL1-BAX) was suspended in 4 ml fresh raff media w/o galactose, respectively. Subsequently, the cultures were grown O/N at 30 °c in the shaking incubator.

E.coli transformation

Team members: Hitesh and Anett

After having been USER ligated, the constructs were ready to be transformed into E. coli.

    Samples:
  1. GAL1-BI1 in W plasmid
  2. TEF1-BI1 in W plasmid

Materials

  • Competent E.coli cells in aliquots of 50 µl
  • USER reacted samples
  • Ice
  • LB media
  • LB plates with Carbecillin (500 µl per 250 ml)

Procedure

  1. Competent cells were collected from the freezer (- 80 °C) and thawed on ice. Each of the tubes should contain 50 µl.
  2. The USER ligated samples (approx. 8 µl) were added to the cells.
  3. The samples were kept on ice for 10 mins.
  4. After incubation on ice, the samples were heat shocked for 30-45 s on the heat block at 42 °C and subsequently briefly cooled down.
  5. Under the clean bench, 1 ml of LB media (w/o antibiotic) was added to each tube.
  6. The bacteria were then recovered for 60 min on the heat block at 37 °C.
  7. After incubation the cultures were span down for 1 min at 22,4 °C and 8000 rcf.
  8. 900 µl of the supernatant was removed from each tube and the pellet resuspended in the remaining media.
  9. Approximately 75 µl of the cultures were then spread on LB plate with Carbenicillin and incubated O/N at 37 °C.

Results

The plates looked really good and had many individual colonies.

3rd October 2019

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

Team members: Noël & Jonas

Materials & Procedure

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

Galactose induction assay of pGAL1-BAX (liquid)

Team members: Noel & Swenja

A galactose induction assay was performed in 24-well plates for the yeast containing the pGAL1-BAX construct. A control was prepared with a yeast strain containing the empty vector.

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. Besides, two wells of each plate were filled with 1 ml raff media w/o galactose.
  6. The OD at 600 nm was measured using a 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.

Galactose induction assay of pGAL1-BAX (solid)

Team members: Swenja & Noel

Next to the liquid assay, a galactose induction assay was performed using agar plates with different concentrations of galactose (0.1%, 0.2 %, 0.3%, 0.4%).

Materials

  • Raff agar w/o galactose, uracil and tryptophan
  • Tryptophan stock (2 g/l)
  • Petri dishes
  • Spatula
  • O/N culture of control (empty vector)
  • O/N culture of pGAL1-BAX
  • Sterile galactose stock (40%; 400 g/l)

Procedure

  1. 0.5 ml of tryptophan stock were added to eight sterile petri dishes, respectively.
  2. To obtain four different galactose concentrations, 250 µl, 188 µl, 125 µl and 63 µl of a 40% galactose stock were added to two plates, respectively.
  3. All plates were then filled with raff agar w/o galactose, uracil and tryptophan until a total volume of 25 ml was reached in each plate (using glass pipettes).
  4. The still liquid agar was immediately mixed with a spatula.
  5. 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.
  6. Subsequently, dilution series were made of both cultures leading up to a dilution of 10-4.
  7. All plates were divided into six sections with a marker.
  8. On one half of the plates, 10 µl of the dilutions 10-2, 10-3 and 10-4 of pGAL1-BAX culture were spotted on the agar. On the other half, 10 µl of the same concentrations of the control culture were spotted.
  9. The plates were then incubated for 3 days at 30 °C.

Results

The results can be seen in the figure below. When bax was expressed, growth was significantly inhibited. Not only were the CFU/ml decreased, but also the colonies were notably smaller in size, indicating that the yeast cells have grown and proliferated at first, but then prematurely died before a normal sized yeast colony could be formed.

Quantitative galactose-induction of the genome integrated pGAL1-BAX. A-D. Both yeast containing pGAL1-BAX or the respective empty vector were grown in different dilutions on raff-U plates with varying galactose concentrations (0.1%, 0.2%, 0.3%, 0.4%). E-F. Close-ups of dilutions 10-2 and 10-3 of yeast expressing pGAL1-BAX. The formed colonies were significantly decreased in size. Some plates showed single colonies on dilution 10-3 while there were no colonies on most plates.

Colony PCR

Team members: Benedicte

Colony PCR was performed for 8 colonies of two constructs.

Materials

  • 8 colonies of the following plates:
    1. pTEF1-BI-1
    2. pGAL1-BI-1

Procedure

  1. A mastermix comprising the following compounds was done. (Volumes per sample):
    1. 4 µl milli-Q water
    2. 0.5 µl of each primer working solution (primers: F=YEA3, R=UBI-1-R, and F=GAL1-F, R=UBI-1-R)
    3. 5 µl 2x MangoMix
  2. Marked colony was picked from plate and transferred into its respective master mix tube.
  3. The samples were put into a PCR machine 2 with the following program:
    1. 96 °C for 30 s
    2. 96 °C for 20 s
    3. 55 °C for 25 s
    4. 72 °C for 1 min 30 s
    5. 72 for 5 min
    6. 12 °C infinite
  4. Steps b. to d. have been repeated for 32 cycles.

Results

As seen in the pictures, positive colonies were identified from TEF-BI1 in colony 2,3,5,8. (length: 1143bp) For GAL-BI1, positive colonies were identified from colony 1,2,4 (length: 1162bp)

The leftside bands correspond to GAL-BI1 from colony 1-8. The right side corresponds to TEF-BI1, colony 1-7.
On the 2nd well TEF-BI1 colony 8 can be seen, and the negative control can be seen in the 4th well (no band).

Inoculation of TEF1-BI-1 and GAL1-BI-1 E. coli

Team members: Jonas and Noël

Colonies chosen for the colony PCR were also inoculated in liquid LB + Amp media for further use.

4th of October

Mini prep - Plasmid DNA purification

Team members: Ojas & Signe

The plasmid purification was performed on the nuclear receptor, the empty pUUS and pWUS plasmids, pWUS-TEF1-BI1 colony 2, 3,5 & 8 and pWUS-GAL1-BI1 colony 1,2 & 4.

Materials

  • E.Z.N.A Plasmid DNA Mini Kit

Procedure

  1. Followed the protocol as described in the kit

Results

The concentration of the purified samples was measured by nanodrop.

Sample No. Sample name Concentrations (ng/uL)
1 Nuclear receptor 344.8
2 pUUS empty 107.2
3 pWUS 41.7
4 pWUS-TEF1-BI1 colony 2 113
5 pWUS-TEF1-BI1 colony 3 67.1
6 pWUS-TEF1-BI1 colony 5 50.5
7 pWUS-TEF1-BI1 colony 8 42.8
8 pWUS-GAL1-BI1 colony 1 43.6
9 pWUS-GAL1-BI1 colony 2 44.1
10 pWUS-GAL1-BI1 colony 4 52.3

USER ligation

Team members: Iben & Jonas

The following fragments were USER ligated

  1. GAL1 + BAX in linearized pUUS vector
  2. TEF1 + BAX in linearized pUUS vector
  3. ADH2 + BAX in linearized pUUS vector
Normal protocol was followed

E. coli transformation

Team members: the ones who must not be named

Normal protocol was followed, and the ligated constructs from above were transformed into competent E. coli

5th of October

Liquid inoculation of E.coli containing GAL1-BAX in pUUS, TEF1-BAX in pUUS, ADH2-BAX in pUUS

Team members: Claudia (and Anett)

Materials and Procedure

The 8-8 cultures that were picked for Colony PCR were inoculated at 37° C in LB+CAR liquid media.

Colony PCR of E.coli containing GAL1-BAX in pUUS, TEF1-BAX in pUUS, ADH2-BAX in pUUS

Team members: Anett (and Claudia)

Materials

  • 10X X7 PCR buffer
  • X7 polymerase
  • F-primer
  • R-primer
  • dNTP
  • Template (colonies from O/N plates)

Procedure

  1. A 3 different MasterMix was created including everything but the template.
  2. 8-8 PCR tubes were filled 9.9 µl of MasterMix.
  3. 8 colonies were chosen and marked for each plate.
  4. Under the sterile bench, the templates were collected by gently picking up as little as possible with a pipette tips.
  5. To suspend all the templates, the samples were mixed by pulling the pipette gently up and down.
  6. The standard PCR protocol was then run.
    1. 98 °C for 30 s
    2. 98 °C for 20 s
    3. 55 °C for 25 s
    4. 72 °C for 2 min 30 s
    5. 72 °C 5 min
    6. 12 °C indefinitely
    b) to d) were run in 30 cycles
Materials Quantity (µl)
10X X7 PCR buffer 1.0
dNTPs 0.8
F-primer (10 µM) 0.5
R-primer (10 µM) 0.5
Template 0.1
X7 polymerase 0.1
mQ water 7.0
Total 10

Results

GAL-BAX colony 1-7 in well 2-8 and TEF1-BAX colony 1-7 in well 10-16
ADH2-BAX colony 1-7 in well 2-8

Miniprep

Team members: Iben

The following samples were positive from the colony PCR performed earlier in the day:

  • GAL1-BAX: colonies 1, 2, 3, 4, 5, 6
  • TEF1-BAX: colonies 2, 3, 4, 5, 6
  • ADH2-BAX: colonies 1, 2, 3, 4, 5, 6, 7
Miniprep was performed on the following samples:
  • GAL1-BAX: colonies 1, 2, 3
  • TEF1-BAX: colonies 2, 3, 4
  • ADH2-BAX: colonies 1, 2, 3

Materials

  • Miniprek kit mentioned previously

Procedure

  1. The protocol from the kit was followed

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_Copenhagen

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