Part:BBa_K649204:Experience

Applications of BBa_K649204

To characterize this part, we constructed the generator part BBa_K649201.

K649201 is expected to express GFP when the lox sites are excised and RFP when they are not.

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	+

We prepared a competent cell JM2.300 into which Pbad/araC-Cre(pSB1A2, BBa_I718008) had been constructed. Subsequently, our BioBrick was constructed into the cell. The strain was grown in a 3ml liquid culture, and 75μl of 2M arabinose was added to induce Cre expression. For the control experiments we used the same strain without arabinose induction and a JM2.300 strain which was induced with arabinose and had only our BioBrick. All the strains were cultured each for periods of 0.5, 1, 2, and 4 hours, and in each case the florescence levels were measured by flow cytometer and FLA.

We measured the fluorescence level to prove that recombination only occurs in presence of Cre recombinase. On the sample with the Pbad/araC-Cre construction, we found that recombination occurred when arabinose was added.

[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 FLA 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.

Green florescence level detected by flow cytometer at period of 1hr.(a)cre-expressing plasmid existing, Ara + (sample 1) (b)no cre-expression, Ara+(sample 3)

Cre-meditated recombination at lox2272 cassette. Period of 0.5hr. (3)The leftmost is a negative control which don't have Cre-expressing plasmid. (1)The center is an arabinose induced sample which has both Cre plasmid and BioBrick BBa_K649201. (2)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.

These results support the fact that this part works.

Recombination frequency of this BioBrick was compared with another lox site carrying part, BBa_K649202(lox71/66). It was found that the excision occured more frequently on BioBrick K649202. To see more about this, please see [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8.1. our work in Tokyo_Tech 2011 wiki].

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