Part:BBa_K4656008

pchA-PpchA-PLEE1-CI

After polymerization of butyrate and Lrp into binary complex, the dimer 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 Cl 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 Cl repressor protein is produced, and if the perceived content of butyrate is low, less Cl 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 Cl 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 sensory module 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.

Then we added an inhibitory protein CI after the genetic route PpchA-pchA-PLEE1-EGFP to reverse the inductive effect of butyrate. We used the components BBa_K4656001, BBa_K4656004, BBa_K4656005, and BBa_K4656006, and designed a gene route PpchA-pchA-PLEE1-Cl-Plam-EGFP. It was cloned into the target vector pET28a(+) and then transformed into the engineered bacteria. Among them, Cl protein was used to bind promoter Plam and inhibit downstream EGFP expression, and the amount of Cl 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 Cl protein production). This once again verified the feasibility of our sensory module -- the increase in butyrate concentration could indeed induce the increase in the expression of the engineered bacteria label protein Cl, that is, it verified that our engineered bacteria could indeed feel the increase in butyrate concentration more sensitively.

Experimental results

1. We introduced the plasmid pet28a-PpchA-pchA-PLEE1-EGFP into E. coli and performed co-culture experiments with different butyrate concentrations. We found that the higher the butyrate concentration, the stronger the fluorescence intensity, demonstrating the feasibility of our route (Figure 1).

Figure1. Fluorescence Intensity(OD480/OD600) with 0mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-EGFP engineered bacteria

a.Fluorescence observation of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic

b.Fluorescence intensity of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate

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2. We then introduced the plasmid pet28a-PpchA-pchA-PLEE1-CI-Plam-EGFP into E. coli to reach a reverse inductive effect of butyrate and performed co-culture experiments with different butyrate concentrations. This time, we found that the higher the butyrate concentration, the lower the fluorescence intensity, in line with our expectations, demonstrating the feasibility of our route (Figure 2).

Figure2. Fluorescence Intensity(OD480/OD600) with 0mM, 5mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria

a.Fluorescence observation of PpchA-pchA-PLEE1-Cl-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic

b.Fluorescence intensity of PpchA-pchA-PLEE1-Cl-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate

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