Composite

Part:BBa_K4656008

Designed by: Xinji Gu   Group: iGEM23_NMU-China   (2023-08-09)
Revision as of 16:27, 16 September 2023 by Juejue (Talk | contribs) (→‎Experimental results)


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.


Usage and Biology

We verified the feasibility of the receptor module pathway, that is, whether engineered bacteria can sense the rise in butyrate concentration. Using components BBa_K4656001, BBa_K4656004, and BBa_K4656005, we designed a gene route PpchA-pchA-PLEE1-EGFP, and cloned it into the target vector pET28a, then transformed the engineered bacteria. The route formed by these components can be used to verify the feasibility of the sensing module. In the experiment, sodium butyrate of 0mM, 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 20mM sodium butyrate had the most obvious promoting effect on fluorescence expression, followed by 10mM sodium butyrate, and the addition of 0mM sodium butyrate had almost no increasing effect on EGFP expression even if the observation time was long. Thus, the feasibility of our receptor module pathway was verified, that is, the increase of butyrate concentration could indeed induce the increase of label protein expression of engineering bacteria, that is, our engineering bacteria could indeed feel the increase of butyrate concentration more sensitively.

Experimental results

Fluorescence Intensity(OD480/OD600) with 0mM, 10mM and 20mM butyrate addition in PpchA-pchA-PLEE1-EGFP:



Fluorescence Intensity(OD480/OD600) with 0mM, 5mM, 10mM and 20mM butyrate addition in PpchA-pchA-PLEE1-CI-Plam-EGFP:



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 143
    Illegal AgeI site found at 609
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 75


[edit]
Categories
Parameters
None