Difference between revisions of "Part:BBa K649201:Experience"
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===Applications of BBa_K649201===
 
===Applications of BBa_K649201===
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We prepared a competent cell JM2.300 into which Pbad/araC-Cre(pSB1A2, BBa_I718008) had been constructed. Subsequently, the 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 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 FLC.
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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
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2. Each cultured medium was 6 times diluted in the medium. From medium of strain having both plasmid, two samples (3ml each) were dispensed and only one side was induced by arabinose (2M, 75 µL). From medium of strain having only one plasmid, 3ml was dispended into a round tube and supplied by arabinose (2M, 75 µL).
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[[Image:table1.png|thumb|center|466px|]]
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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.
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4. In the same way as 3, 9 more samples were examined at period 1hr, 2hr, and 4hr.
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We measured the fluorescence levels 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. We could also observe recombination occurred when arabinose was not added, which can be explained due to a leaking in the Pbad/araC promoter. Opposite to this result, when we measured the levels of the sample without the Pbad/araC-Cre construction, we found that the GFP levels were far lower than those of the sample with the Pbad/araC-Cre construction. This clearly proves that our lox constructions respond correctly to the effects of Cre recombinase.
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We also confirmed our results optically by taking florescence photographs. Whole samples were plated and incubated in 37℃ for 10 hours. Photographs of three samples of BBa_K649201 were taken at t = 0.5 hours using red florescence filter, green florescence filter and no filter as shown below, respectively.
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===User Reviews===
 
===User Reviews===

Revision as of 21:16, 3 October 2011

Applications of BBa_K649201

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. From medium of strain having both plasmid, two samples (3ml each) were dispensed and only one side was induced by arabinose (2M, 75 µL). From medium of strain having only one plasmid, 3ml was dispended into a round tube and supplied by arabinose (2M, 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.

User Reviews

UNIQ306f40806273cd54-partinfo-00000000-QINU UNIQ306f40806273cd54-partinfo-00000001-QINU