Team:UCopenhagen/Notebook/killswitch/week33

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Kill Switch Week 33 (12th-18th of August)

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12th August 2019

1. PCR amplification of BI-1, GAL1, TEF1 and ADH2

Team members: Swenja and Noël

Materials

• Template
• X7 polymerase
• Primers
• dNTPs
• HF buffer (5X)
• MilliQ water

Procedure

1. Normal protocol for PCR was followed with the following conditions:
2. PCR program 30 cycles of:
   1. 98 °C: 30 seconds
   2. 98 °C: 20 seconds
   3. 55 °C: 25 seconds
   4. 72 °C: 2 minutes and 30 seconds
   5. 72 °C: 5 minutes
   6. 12 °C: Infinity

Results

All sequences got amplified

13th of August 2019

1. Gel electrophoresis of PCR products

Team members: Swenja

To confirm the constructs, the PCR samples from the 12th of August were run in a gel.

Materials

• 1 % agarose
• 1X TAE-buffer
• Loading dye
• DNA ladder
• PCR tubes
• Ice box

Procedure

1. To prepare the gel:
   1. 1% agarose gel was taken from the common stock in electrophoresis room
   2. The Agarose-TAE buffer solution was poured into a labelled casting tray.
2. Setting up the electrophoresis chamber:
   1. Solidified gel was placed on the electrophoresis chamber filled with TAE buffer (after 5 uses).
   2. To prepare the sample to be loaded, 4 μL of each sample was added into a new PCR tube and mixed with 1 μL of loading dye.
   3. To load the wells, the second well was filled with 1 kb DNA ladder while wells 3-6 were loaded with the PCR samples (1-4). In the fifth well a negative control was loaded.
   4. Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 15-20 mins.

Data

Sample
1. BI-1
2. GAL1
3. TEF1
4. ADH2

Results

2. Plasmid digestion for USER ligation

Team members: Swenja

As the template for the promoters (pGAL1, pTEF1, pADH2) obtained from the instructors, Simon and Victor, were in a plasmid. Therefore, a digestion step was performed with DpnI enzyme before proceeding with the USER ligation.

Materials

• Dpn1
• Amplified samples

Procedure

1. 0.5 µl have been added to the PCR samples, respectively. After that they were incubated for 1 h at 37 °C.

Sample
GAL1
TEF1
ADH2

3.PCR clean up

Team members: Swenja & Anett

The PCR products were purified/cleaned up using the E.Z.N.A Cycle Pure Kit.

Materials

• E.Z.N.A. Cycle Pure Kit
• 1.5 ml eppendorf tubes

Procedure

1. 40 µl of all the PCR samples as mentioned in the Table below were transferred into a 1.5 mL microcentrifuge tube.
2. 200 µl of the CP buffer was added.
3. The samples were vortexed and centrifuged briefly.
4. HiBind DNA mini columns were inserted into a 2 mL collection tube.
5. The samples from step 3 were added to the HiBind DNA mini column.
6. Centrifugation was carried out at 13000 g for 60 seconds at room temperature. The filtrate was discarded and the collection tubes were reused.
7. 700 µl of DNA wash buffer diluted with 100% ethanol was added. Centrifugation was carried out at maximum speed for 60 seconds. The filtrate was discarded and the collection tubes were reused.
8. Step 7 was repeated again for a second DNA wash buffer step.
9. The empty HiBind DNA mini columns were centrifuged at maximum speed for 2 minutes to dry the column. This step was critical to remove the trace ethanol.
10. The HiBind DNA mini column was transferred into a new 1.5 mL microcentrifuge tube.
11. 50 µl of elution buffer was directly added to the center of the column matrix. It was kept at room temperature for 2 minutes and was centrifuged at maximum speed for 60 seconds and then put on ice.
12. The concentrations were then measured using NanoDrop.
13. The samples were stored at 20 °C.

Sample	DNA concentration
BI-1	47.4 ng/µl
GAL1	49.0 ng/µl
TEF1	53.4 ng/µl
ADH2	35.2 ng/µl

15th August 2019

1. USER ligation

Team members: Swenja and Noël

USER ligation was performed to create the following constructs:

Sample
1. XI2A + pTEF1 + BAX
2. XI2A + pADH2 + BAX
3. B + pGAL1 + BI-1
4. XI2A + pGAL1 + BAX

The plasmids containing the promoters had been previously digested on the 13th of August.

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

Compound	Amount (µl)
USER enzyme	1
CutSmart buffer	1
Promoter	1
Gene	1
Vector backbone	1
Water	5
Total	10

Results

The wrong (undigested) vectors had been used why the USER ligation has been successfully repeated later on the same day.

2. E. coli transformation

Team members: Swenja and Hitesh

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

Sample
1. XI2A-pTEF1-BAX
2. XI2A-pADH2-BAX
3. B-pGAL1-BI-1
4. XI2A-pGAL1-BAX

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.

16th August 2019

1. Colony PCR

Team members: Swenja & Noël

Colony PCR was performed of the transformed E. coli cells from the 15th of August.

Sample
1. XI2A + pTEF1 + BAX
2. XI2A + pADH2 + BAX
3. B + pGAL1 + BI-1
4. XI2A + pGAL1 + BAX

Materials

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

Procedure

1. A MasterMix was created including everything but the template and the primers.
2. 10 wells of a 96-well PCR plate were filled with the respective F- and R-primers for each of the 4 samples. (S1 A1-10, S2 B1-10, S3 C1-10, S4 D1-10)
3. After that 8.9 µl of MasterMix was added to each well.
4. 10 colonies were chosen and marked for each plate.
5. Under the sterile bench, the templates were collected by gently picking up as little as possible with a pipette tips.
6. The tips were put into the matching wells and left until all templates had been collected. (NOTE: Following samples were mixed up - A1 + A9, A4 + A7, B4 + B7, D8 and D9)
7. To suspend all the templates, the samples were mixed by pulling the pipette gently up and down.
8. 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
   7. b) to d) were run in 30 cycles

Materials	Quantity (µl)
10X X7 PCR buffer	1
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
Total	10

Sample	F-primer	R-primer
1. XI2A + pTEF1 + BAX	YEA3	U-Bax-R
2. XI2A + pADH2 + BAX	YEA21	U-BI-1-R
3. B + pGAL1 + BI-1	Gal FP	U-Bax-R
4. XI2A + pGAL1 + BAX	Gal FP	U-Bax-R

Results

Unfortunately, the gel images were all blurry. Another Colony PCR using MangoMix instead of X7 PCR buffer was conducted the same day.

18th August 2019

1. Repetition of Colony PCR

Team members: Swenja and Ojas

Colony PCR was repeated for the transformed E. coli cells from the 15th of August.

Samples
1. XI2A + pTEF1 + BAX
2. XI2A + pADH2 + BAX
3. B + pGAL1 + BI-1
4. XI2A + pGAL1 + BAX

Materials

• 2x MangoMix
• F-primer
• R-primer
• Template (colonies from O/N plates)

Procedure

1. A MasterMix was created including everything but the template and the primers.
2. After that 8.9 µl of MasterMix was added to each well
3. 10 wells of a 96-well PCR plate were filled with the respective F- and R-primers for each of the 4 samples. (S1 A1-10, S2 B1-10, S3 C1-10, S4 D1-10)
4. 10 colonies were chosen and marked for each plate.
5. Under the sterile bench, the templates were collected by gently picking up as little as possible with a pipette tips.
6. The tips were put into the matching wells and left until all templates had been collected.
7. To suspend all the templates, the samples were mixed by pulling the pipette gently up and down.
8. The following PCR protocol was then run.
   1. 96 °C for 30 s
   2. 96 °C for 20 s
   3. 55 °C for 25 s
   4. 72 °C for 2 min 30 s
   5. 72 °C 5 min
   6. 16 °C indefinitely
   7. b. to d. were run in 35 cycles

Materials	Quantity (µl)
2x MangoMix	5
F-primer (10 µM)	0.5
R-primer (10 µM)	0.5
Template	0.1
mQ water	4
Total	10

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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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