Difference between revisions of "Part:BBa K4656008"
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<html><BR><BR><center><img style="display: block;-webkit-user-select: none;margin: auto;background-color: hsl(0,0%,90%);transition: background-color 300ms;" src="https://static.igem.wiki/teams/4656/wiki/check-in-2.png"></center> | <html><BR><BR><center><img style="display: block;-webkit-user-select: none;margin: auto;background-color: hsl(0,0%,90%);transition: background-color 300ms;" src="https://static.igem.wiki/teams/4656/wiki/check-in-2.png"></center> | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
1. Verify the feasibility of the receptor module pathway, that is, verify whether engineered bacteria can sense the rise in butyrate concentration. | 1. Verify the feasibility of the receptor module pathway, that is, verify whether engineered bacteria can sense the rise in butyrate concentration. | ||
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https://static.igem.wiki/teams/4656/wiki/sensor2-result.png | https://static.igem.wiki/teams/4656/wiki/sensor2-result.png | ||
<p>sensor2 Fluorescence Intensity(OD480/OD600)</p> | <p>sensor2 Fluorescence Intensity(OD480/OD600)</p> | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K4656008 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4656008 SequenceAndFeatures</partinfo> |
Revision as of 14:02, 6 September 2023
pchA-PpchA-PLEE1-CI
After polymerization of butyrate and Lrp into binary complex, it can bind to PpchA promoter, thus activating the transcription and expression of pchA. The generated pchA can bind to the promoter PLEE1, thereby activating downstream CI repressor protein expression. This short genetic route can be used as a sensing module to sense the amount of butyrate in the gut. If the perceived content of butyrate in the gut is high, more CI repressor protein is produced, and if the perceived content of butyrate is low, less CI repressor protein is produced. By combining this gene route with the gene route of BBa_K4656007, when adding EGFP to the TPH1 and TDC of BBa_K4656007, the content of CI repressor protein can be determined by detecting fluorescence and then the content of butyrate in the intestine can be calculated. In addition, the protein expression of Plam-TPH1-TDC1 gene route can also be determined by special methods to detect 5-HT content in the intestine, and then the content of butyrate in the intestine can be estimated.
![](https://static.igem.wiki/teams/4656/wiki/check-in-2.png)
sensor1(0)
https://static.igem.wiki/teams/4656/wiki/sensor1-10.pngsensor1(10)
https://static.igem.wiki/teams/4656/wiki/sensor1-20.pngsensor1(20)
https://static.igem.wiki/teams/4656/wiki/sensor1-result.pngsensor1 Fluorescence Intensity(OD480/OD600)
2. Reverse thinking to verify the feasibility of the receptor module pathway We used the components BBa_K4656001, BBa_K4656004, BBa_K4656005, BBa_K4656006, and designed a gene route PpchA-pchA-PLEE1-CI-Plam-EGFP. It was cloned into the target vector pET28a(+) and then transformed into the engineered bacteria. Among them, CI protein was used to bind promoter Plam and inhibit downstream EGFP expression, and the amount of CI protein produced was reflected by fluorescence intensity. In the experiment, sodium butyrate of 0mM, 5mM, 10mM and 20mM was added respectively, and the fluorescence intensity (OD480/OD600) was measured at 0, 4, 8, 12, 16, 20, 24 and 28h, respectively. We found that the fluorescence expression at 0mM sodium butyrate was the largest, and that at 20mM sodium butyrate, even if the observation time was long, there was almost no effect of increasing the fluorescence expression, and the higher the concentration of sodium butyrate, the more significantly the fluorescence expression was inhibited (indicating the more CI protein production). This once again verified the feasibility of our receptor module pathway -- the increase in butyrate concentration could indeed induce the increase in the expression of the engineered bacteria label protein CI, that is, it verified that our engineered bacteria could indeed feel the increase in butyrate concentration more sensitively. https://static.igem.wiki/teams/4656/wiki/sensor2-0.pngsensor2(0)
https://static.igem.wiki/teams/4656/wiki/sensor2-5.pngsensor2(5)
https://static.igem.wiki/teams/4656/wiki/sensor2-10.pngsensor2(10)
https://static.igem.wiki/teams/4656/wiki/sensor2-20.pngsensor2(20)
https://static.igem.wiki/teams/4656/wiki/sensor2-result.pngsensor2 Fluorescence Intensity(OD480/OD600)
Sequence and Features