Part:BBa_K649201:Experience

Applications of BBa_K649201

sample		arabinose
1	PlacIQ-lox2272-RFP-lox2272-GFP(pSB3K3) PBAD/araC-Cre(pSB1A2)	+
2	PlacIQ-lox2272-RFP-lox2272-GFP(pSB3K3) PBAD/araC-Cre(pSB1A2)	-
3	PlacIQ-lox2272-RFP-lox2272-GFP(pSB3K3)  : negative control	+

[Method]
1. Strain JM2.300 with both Pbad/araC-Cre and PlacIQ-lox2272-RFP-lox2272-GFP in it was cultured at 37℃ in 3ml of LB medium containing ampicillin (6 µL) and kanamycin(3.6 µL). Same kind of strain having only PlacIQ-lox2272-RFP-lox2272-GFP was cultured in same volume of LB containing kanamycin (3.6 µL) and carbenicillin(4 µL). These were cultured until OD 1.6.

2. Each cultured medium was 6 times diluted in the medium and three samples (3 ml each) were dispensed from those. Among them, two were induced by arabinose (2 M, 75 µL).

3. After 30 min from induction, 1000 times dilution and 100000 times dilution of each sample were plated and incubated in 37°C about 12 hours. The florescence of plates was examined by FLC and it was taken picture. About the rest of the samples, strains were harvested by centrifugation and suspended by adding 1 mL of PBS (phosphate-buffered saline). The last OD of PBS solution was approximately 0.4. We dispensed 700 µL of each suspension into a disposable tube through a cell strainer, and fluorescence intensity of each cell was measured with a flow cytometer of Becton, Dickinson and Company.

4. In the same way as 3, 9 more samples were examined at period 1hr, 2hr, and 4hr.

Effect of Cre-meditated recombination at lox2272 cassette. The leftmost is a negative control which don't have Cre-expressing plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick BBa_K649201. The rightmost is a uninduced strain which has both plasmid like as the center.

Detection of GFP
The order of samples is same as above.

Detection of mCherry Overlay
The order of samples is same as above.

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