Difference between revisions of "Part:BBa K4442001"

Revision as of 10:30, 6 October 2023

The butyrate sensing system PpchA-PLEE1 originates from the enterohemorrhagic Escherichia coli strain O157:H7 which was discovered by Nakanishi et al. (2009). The system below includes the sequences of the pchA promoter and pchA regulator, located upstream, as well as the LEE1 promoter and RBS downstream. A complex of the leucine-responsive regulatory protein and butyrate activates the system. To add an fluorescent output or any other sort of output protein, simply add the base pairs downstream of the RBS.

Nakanishi et al.1 found that the leucine-responsive regulatory protein (Lrp) forms a complex with butyrate which after binding to the promoter of pchA, activates the transcription of the pchA regulator. The pchA regulator then binds to the promoter of LEE1, to transcribe the ler gene. In Bai & Mansell’s2 paper, the ler gene was replaced with GFP to report the sensing of butyrate. Similarly, in our project, the pchA regulator would bind to the promoter of LEE1 but instead express YFP.

More information can be obtained here: Nakanishi N, Tashiro K, Kuhara S, Hayashi T, Sugimoto N, Tobe T. Regulation of virulence by butyrate sensing in enterohaemorrhagic escherichia coli. Microbiology. 2009;155(2):521–30. Bai Y, Mansell TJ. Production and sensing of butyrate in a probiotic E. coli strain. International Journal of Molecular Sciences. 2020;21(10):3615.

A decrease in butyrate concentration in fecal samples was found to correlate with depression. As part of our goal to create a microbial sensor to aide in the diagnosis of depression, this system will allow us to detect the levels of butyrate through fluorescence intensity.

The pchA promoter, pchA, and LEE1 promoter sequences were all obtained from the original study authors, Yanfen Bai and Thomas J. Mansell.

NMU_China_2023

Correction

The team got the wrong order of the composite sequence in mistakes, which should be Ppcha-pcha-plee1 actually.

Short description

The Butyrate Sensing System can "sense" the level of butyrate, its core event is the binding of the Lrp-butyrate dimer complex to the promoter PpchA. Since the activation of promoter PpchA can promote downstream gene expression, the volume of downstream products can reflect the perceived butyrate concentration.

Our validation via experiments

Our team has made the following additions to the development and validation of the Butyrate Sensing System:

1. We designed two gene segments: PpchA-pchA-PLEE1-EGFP and PpchA-pchA-EGFP. After characterization, we measured and compared fluorescence intensity and bacterial growth curves, proved that the segment involving plee promoter (i.e. PpchA-pchA-PLEE1-EGFP) has a higher sensitivity to butyrate as well as having a stronger ability to enhance downstream gene expression.

The purple line shows PpchA-pchA-PLEE1-EGFP, while the yellow line shows PpchA-pchA-EGFP.

2. We demonstrated in controlled experiments that increased Lrp expression can indeed enhance the sensitivity of PpchA to butyrate. <p>

The purple line shows extra Lrp expression, while the yellow line shows normal Lrp expression.

3. We tested the capacity for "sense" of the designed gene segment PpchA-pchA-PLEE1-EGFP, and found that the higher the concentration of butyrate, the stronger the fluorescence, which proved that our engineered bacteria could indeed sensitively sense the increase of butyrate concentration. Then, we designed the gene segment PpchA-pchA-PLEE1-CI-Plam-EGFP, in which CI protein was used to bind promoter Plam and inhibit downstream EGFP expression. The experiment found that the higher the concentration of butyrate, the more obvious the inhibition of fluorescence expression (indicating that the more CI protein produced by butyrate). This confirmed once again that our engineered bacteria could indeed sensitively sense an increase in butyrate concentration. Overall, we validated the feasibility of butyrate receptors.

<p>Fig. 2 |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

@@ Line 31: / Line 31: @@
 <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/sensor1.jpg"width="700" height="650"></center></html>
-           <b>Fig. 2 |</b>Fluorescence Intensity(OD480/OD600) with 0mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-EGFP engineered bacteria
+           <p><b>Fig. 2 |</b>Fluorescence Intensity(OD480/OD600) with 0mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-EGFP engineered bacteria.</p>
-             <p>a.Fluorescence observation of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic</p>
+             <p> a.Fluorescence observation of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic</p>
             <p> b.Fluorescence intensity of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate</p>