Part:BBa_K2120302
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 1790
Illegal AgeI site found at 1902 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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.
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