Part:BBa_K2120302
araC-pBAD+B0034+RFP+B0015
pBAD is a promoter induced by arabinose and arac is the repressor. it can be used to control the expression of our reporter gene RFP, which indicate the strength of promoters
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 BBa_K808000 and we connect BBa_J100012 downstream. When the promoter is induced by arabinose, the transcription of the RFP will begin. And the expression of RFP will be depended on the concentration of arabinose added. In a narrow range of arabinose concentration, there is a liner relationship between expression and induction.
Usage and biology
Inducer: L(+)-arabinose We designed this biobrick to simulate the expression of the inhibitor in BBa_K2120310 and BBa_K2120311. And we hope to know the expression level of downstream gene when we added the same concentration of arabinose as K2120310 and K2120311.
Test
We transformed the plasmids, pSB1C3, contained this device into E.coli TOP 10. And we tested it in LB medium with different arabinose concentration. We add 2ml activated bacteria into new LB medium and induce by adding arabinose. The negative control is pSBIC3 without K2120302. The positive control is the bacteria contained K2120302 but no arabinose. And we measured the fluorescence intensity and OD600 at the same time. The fluorescence measurement is carried by Bioteke plate reader. Then we get the single cells’ fluorescence intensity by fluorescence intensity being divided by OD600.
Result
We repeated this test twice. 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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