4 agar plates with LigA = 2 for irradiation and 2 for control.
4 liquid cultures with LigA = 2 for irradiation and 2 for control.
4 agar plates with = 2 for irradiation and 2 for control.
4 liquid cultures with = 2 for irradiation and 2 for control.
There are two groups, each with 8 cultures.
Shift one: 2 agar plates and 2 liquid cultures with LigA and 2 agar plates and 2 liquid cultures with were put in 16°C for one hour. After an hour, half of them were irradiated with blue light for an hour while the other have was wrapped in aluminium foil as a control. Afterwards they are put in the 37°C incubator for some time
Shift two: they were put in the 37°C for some hours so that the cultures can grow.
After measurement of the first shift the following was concluded:
Although the cells with had a small signal after FACS, the plates showed no GFP when put under the lamp.
LigA that was not exposed to blue light had the same amount of signal as the construct that had been exposed. we assume that this is due to the exposure to white light when the cultures are exposed. Possibly, the promotor was then already activated since white light contains blue light frequencies. A new set up will be made over the weekend where all cultures will be made in a dark room.
Dilutions of the shift one liquid cultures were made + 2 new cultures from the motherplate from ligA. These were put overnight under blue light together with the cultures of shift 1
Since we want to avoid gel purification, we use the direct sample from the restriction digest
The restriction enzymes are denatured by keeping the sample at 85°C for 20 minutes
vector
insert
Terminator
SAMS
2000 bp
3070 bp
50 ng
280 ng
10 μl
16 μl
The genes fcs and ech are individually cut with the four restriction enzymes
EcoR1
fcs RD-E
Spe1
fcs RD-S
Pst1
fcs RD-P
Xba1
fcs RD-X
EcoR1
Ech RD-E
Spe1
Ech RD-S
Pst1
Ech RD-P
Xba1
Ech RD-X
Results on gel show that all the genes have the same length and display 1 line.
SAMS I A RD is put on gel, to be used for gel extraction and a part of SAMS I B RD is put on gel to check the length.
No signal... grrr...
Replated colonies from the original SAMS plate from 24/8
Took 4 individual colonies and plated them on 4 corners of a petri dish
the 4 genes(sam5, sam8, ech, fcs) where taken from the original DNA that was sent, electroporated and plated. This in order to make a glycerol stock.
We also started another method to try and ligate the different genes for vanillin production by using PCR. Today we did PCR on the ech, fcs, sam5 and sam8 genes to get the DNA out of the plasmid. We used the prefix and suffix primers from last year, these primers keep the pre- and suffix of the biobrick part intact and allow us to work with the purified biobricks themself.
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