Difference between revisions of "Part:BBa K2120302"
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<partinfo>BBa_K2120302 short</partinfo> | <partinfo>BBa_K2120302 short</partinfo> | ||
| − | 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 | + | 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 |
| Line 22: | Line 22: | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
| − | This device contains [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808000 BBa_K808000] and we | + | This device contains ''araC''-pBAD([https://parts.igem.org/wiki/index.php?title=Part:BBa_K808000 BBa_K808000]) and we ligate it with the inhibitor of pR promoter, ''cI''([https://parts.igem.org/wiki/index.php?title=Part:BBa_J100012 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''' | '''Usage and biology''' | ||
Inducer: L(+)-arabinose | Inducer: L(+)-arabinose | ||
| − | We designed this biobrick to | + | We designed this biobrick to imitate the expression of the inhibitor in our two threscold test circuits, ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K2120310 BBa_K2120310]) and ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K2120311 BBa_K2120311]). And we aim to test the expression level of downstream gene when concentration of arabinose in culture are same. |
'''Test''' | '''Test''' | ||
| − | We transformed the plasmids, pSB1C3, contained this device into E.coli | + | 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 fluorescence intensity and OD600 at the same time | + | 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''' | '''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. | |
[[File:BIT-CHINA-PARTS-INHIBITOR-1.jpg|600px|thumb|center]] | [[File:BIT-CHINA-PARTS-INHIBITOR-1.jpg|600px|thumb|center]] | ||
[[File:BIT-CHINA-PARTS-INHIBITOR-2.jpg|600px|thumb|center]] | [[File:BIT-CHINA-PARTS-INHIBITOR-2.jpg|600px|thumb|center]] | ||
Revision as of 18:06, 20 October 2016
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 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.
