Difference between revisions of "Part:BBa K2120302"

Revision as of 15:29, 22 October 2016

araC-pBAD+B0034+RFP+B0015

This device is composed of induced promoter pBAD and its inhibitor araC(BBa_K808000), strong RBS B0034, report gene RFP and double terminator B0015. The fluorescence intensity will decrease corresponding the increasing of inhibitor to indicate the switch’s expression level.

Sequence and Features

Functional Parameters

This device contains araC-pBAD(BBa_K808000) and we ligate it with the inhibitor of pR promoter, cI(BBa_J100012) downstream. When the promoter pBAD is induced by arabinose, the reporter gene RFP start transcription. And the expression of RFP will depend on the concentration of arabinose. In a narrow range of arabinose concentration, there is a liner relationship between induction of promoter and expression of reporter.

Usage and biology

Inducer: L(+)-arabinose We designed this biobrick to imitate the expression of the inhibitor in our two threscold test circuits, (BBa_K2120310) and (BBa_K2120311). And we aim to test the expression level of downstream gene when concentration of arabinose in culture are same.

Test

We transformed the plasmids, pSB1C3, contained this device into E.coli BMTOP10. And we tested it in LB medium with gradient arabinose concentration. The negative control is bacteria containing pSB1C3 instead of K2120302. The positive control is the bacteria containing K2120302 but no arabinose.

And we measured the total fluorescence intensity and OD600 at the same time. Then we divide total fluorescence intensity by OD600 to measure the single cell’s fluorescence intensity. We use this value to indicate the expression level of inhibitor .

Result

At the beginning of the growth, cells do not show different fluorescence strength until 5-6 hours. We found the OD600 is preserved at 2.0-2.5 when cell grew for 4-5 hours after induced. In a narrow range concentration, the pBAD shows a different strength. The result of twice test is showed in Fig 1 and Fig 2.

But the liner relationship is not as our expectation. Fig 3 and Fig 4 show a curve. The expression of RFP is similar in twice test.

According to the Fig 1 and Fig 2, the RFP expression level from different test are not similar. But in twice test we can know the arabinose concentration at 0.003% and 0.004% which we got from the test of K2120310 shows a similar tendency. So we regard the 0.004% as the switch threshold for CI-Pr combination. The expression of RFP at 0.003%, 0.004% shows in Fig 5 and Fig 6.