Kill Switch Week 34 (19th-25th of August)

19th August 2019

1. Gel electrophoresis of colony PCR products

Team members: Noël and Swenja

A gel was made for confirmation of positive colonies of the previously performed E. coli colony PCR.

Results

Blue: pTEF-BAX; Orange: pADH-BAX; Yellow: pGAL1-B1; Green: pGAL1-BAX. The following colonies were deemed positive and inoculated for Sequencing:

pTEF-BAX: 1, 2, 3, 4

pADH-BAX: 1, 3, 6, 8

pGAL1-B1: 5, 8, 9, 10

pGAL1-BAX: 1, 2, 3, 4

2. Plasmid purification

Team members: Iben, Swenja & Noël

The plasmids of the positive E. coli colonies that have been inoculated on the 18th of August have been purified using the E.Z.N.A. Plasmid DNA Mini Kit I.

Materials

E.Z.N.A. Plasmid DNA Mini Kit I
O/N cultures of positive E. coli colonies

Procedure

The inoculated cultures were taken from the 37 °C incubator.
The miniprep kit was found in R132: E.Z.N.A. Plasmid DNA Mini Kit I. (For solution I check the fridge in R132)
After mixing, 2 ml of each culture were added into labelled eppendorf tubes.
The samples were centrifuged at 10 000 g for 1 min at RT.
The supernatant was discarded and 300 µl of Sol I added.
The samples were then vortexed and transferred into new microcentrifuge tubes.
We added 300 µl Sol II, inverted the samples and waited for 2-3 minutes (timing is important!)
Subsequently, 400 µl Sol III were added and the tubes inverted until white lines were formed inside the tube.
The samples were spun at maximum speed for 10 minutes.
The supernatants were transferred into columns in 2 ml collection tubes.
The vacuum sucker from Karel’s lab was used to suck from step 8 of the protocol onwards.
700 µl DNA Wash Buffer was added and the columns put into clean collecting tubes after the liquid had been sucked out.
To dry the column it was centrifuged for 2 min at maximum speed at RT.
The columns were then transferred into new microcentrifuge tubes.
80 µl Elution Buffer was added and after 1 min the tubes were centrifuged for 1 min at maximum speed at RT.
The DNA concentrations were then measured with NanoDrop and the samples stored at - 20 °C.

Results


Sample	DNA concentration (ng/µl)
1.1	77.7
1.2	66.6
1.3	75.9
1.4	51.1
2.1	55.5
2.3	99.9
2.6	72.9
2.8	63.1
3.5	51.7
3.8	64.4
3.9	84.6
3.10	69.6
4.1	42.8
4.2	108.7
4.3	62.2
4.4	83.4

Sequencing

Team members: Iben

The following samples were sent for sequencing


No.	Sample	Colonies	Primer 1	Primer 2
1	TEF1-BAX	1.1 & 1.3	YEA74	YEA81
2	ADH2-BAX	2.3 & 2.8	YEA74	YEA81
3	GAL1-BI-1	3.9 & 3.10	YEA76	YEA75
4	GAL1-BAX	4.2 & 4.3	YEA74	YEA81

Results

Colony 4.3: 1 mismatch in GAL1
Colony 2.8: 2 bp gap in promoter

21st August 2019

1. Preparations for yeast transformation

Team members: Iben, Swenja & Noël

To prepare for the upcoming yeast transformation, reagents were checked, plates poured, backbones purified and a culture of yeast inoculated.

Materials

Vector purification: XI2C backbone & ? kit
Preparing plates: Glu-U Agar & Sterile petri dishes
PLI: 1 M Lithium acetate, mqH2O & 50 % PEG
Inoculation of yeast culture: Yeast seed culture (Plate in fridge) & YPD media

Procedure

Vector purification: The ? kit was found in the PCR room and the instructions followed. The DNA concentration was measured using NanoDrop. → XI2C: 46.6 ng/µl
Pouring of Glu-U plates: The Glu-U agar was heated in the microwave until fluid and subsequently cooled down until touchable. The agar was then poured into sterile petri dishes. (Bit more than for E. coli)
Preparation of PLI: 1 ml of 1 M LiAc, 1 ml H2O and 8 ml of 50 % PEG were mixed in a falcon tube.
Inoculation of yeast culture: One colony of the seed culture was picked and resuspended in 5 ml YPD media. The culture was incubated at 30 °C in the shaking incubator O/N.

22nd August 2019

1. Plasmid digestion

Team members: Swenja

The parts for the different assembler systems were mixed and the plasmids digested for later use in the yeast transformation. This step was carried out in the 4 h incubation time of the yeast transformation protocol

Materials

Gene constructs
Empty backbones
CutSmart buffer
Enzyme Not1

Procedure

The needed amounts in µl of each construct had to be calculated. For the 3-assembler system we want 600 ng of the construct in the first backbone (XI2A) and 900 ng for the two other constructs/empty vectors to be added.
The constructs/empty vectors were then mixed together in a PCR tube according to the table below.
The total volume of each tube was calculated and the amount of CutSmart buffer responding to 1/9 of this volume added.
Lastly, 1.5 µl of Not1 enzyme was added to each tube and the content mixed and briefly span down.
The samples were then placed in a PCR machine with the following settings (YTdigest):
1. 37 °C for 2 h
2. 65 °C for 20 mins
3. 12 °C indefinitely


Assembler system nr.	XI2A module	µl	B module	µl	XI2C module	µl	Total volume (µl)
1.	pTEF1-BAX	7.7	pGAL1-BI-1	10.6	XI2C	19.3	37.6
2.	pADH2-BAX	9.5	pGAL1-BI-1	10.6	XI2C	19.3	39.4
3.	pGAL1-BAX	5.5	B	26.5	X12C	19.3	51.3


Sample nr.	Total volume (µl)	Buffer added (µl)	Not1 added (µl)
1.	37.6	4.2	1.5
2.	39.4	4.4	1.5
13.	51.3	5.7	1.5

2. Yeast transformation

Team members: Swenja & Jonas

Yeast transformation was performed all three constructs.

Materials

O/N yeast culture
YPD media
Cuvettes
Salmon sperm
Lithium acetate
Assembler systems
mqH2O
Glu-U agar plates
Gal raff agar plates


Assembler system nr.	XI2A module	B module	XI2C module
1.	pTEF1-BAX	pGAL1-BI-1	XI2C
2.	pADH2-BAX	pGAL1-BI-1	XI2C
3.	pGAL1-BAX	B	X12C

Procedure

0.9 ml YPD media and 0.1 ml of the O/N culture were mixed in a cuvette and the OD(600nm) was measured (YPD as blank).
The culture was then diluted to an OD(600nm) = 0.25 based on the OD value measured (OD of cuvette sample was 0.568, so OD of culture was 5.68. For that 1.1 ml of culture was mixed with 23.9 ml YPD media (use the same as for initial inoculation) in a 50 ml Falkon tube to get a total of 25 ml.
The culture was then incubated for another 4 h to reach an OD(600nm) of approximately 1 (ours had 0.862).
The ssDNA (salmon sperm) was boiled for 10 mins at 99 °C in the heat block and subsequently kept on ice.
The culture was spun down in the Falkon tube at 3000 rcf for 10 mins at RT.
The supernatant was poured into a waste tube with one movement and the pellet resuspended in 1 ml of LiAc.
The culture was then transferred into 1.5 ml eppendorf tubes and span down at 7000 rcf for 1 min at RT.
The supernatant was carefully removed with a pipette and the pellet resuspended in a 100 µl of 0.1 M LiAc.
30 µl of salmon sperm were added and everything was mixed gently.
Subsequently, 1 ml of PLI was added and mixed well.
The DNA samples (see table above) were transferred into 1.5 ml eppendorf tubes and to each sample 200 µl of the yeast culture were added and well mixed.
The samples were then heat shocked for 30 mina at 42 °C in the heat block.
After transformation, the cells were spun down at 7000 rcf for 1 min at RT and the supernatant discarded.
The pellet was resuspended in 1 ml mqH2O.
The cells were spun down at 8000 rcf for 1 min at RT.
900 µl of the supernatant/water were removed and the pellet once again resuspended (gently on lowest vortex level).
Finally, the transformed cells were added onto Glu-U (sample 3) or Gal raff (sample 1-2) plates and distributed with a spatula until the liquid had dried.
The plates were then incubated for 3 nights at 30 °C in the second floor.

Results

There was good growth for the yeast with the assembler system 3 which has BAX under an inducible promoter. For the construct with constitutively produced BAX, there was no growth when the strong promoter (TEF1) was used and some growth but small colonies when the moderate promoter was used (AHD2).

We could think about repeating the experiment with adding galactose to the transformation period of 30 mins. Furthermore, we could consider ordering the ADH1 promoter as both constitutive promoters seems to be too strong to be balanced out by Gal1.