Difference between revisions of "Part:BBa K4656004"

Revision as of 13:18, 22 September 2023

PLEE1

PLEE1 is the promoter of the locus of enterocyte effacement which binds to pchA.

Since the specific binding site of pchA and PLEE1 is still unknown, at present, researchers have purchased Escherichia coli genome from atcc and amplified PLEE1 with different fragments by PCR as candidates, and constructed 6 different kinds of plasmids. +1 is the transcription start site TSS.

"Additionally, the best performing plasmid, pC4S-P4, contains a small part of the ler gene in the PLEE1 promoter region."[1] {The pC4S-P4: pDMB,PpchA-pchA-PLEE1(-98+218bp)-gfp;CmR}

Usage and Biology

The Plee promotor is under the control of PchA, we designed and compared Ppcha-pcha-Plee-egfp and Ppcha-pcha-egfp circuit.

Experiment results

We designed two gene fragments as mentinoned: one directly attaching EGFP downstream of the pchA promoter, and the other appending EGFP after the existing gene segment in enterohemorrhagic E. coli. We will compare the responsiveness and feedback levels of these two fragments to butyrate. We cloned the synthesized A segment and B segment onto the pet-28a plasmid. Subsequently, we will individually transform these plasmids into Escherichia coli BL21. After plating and overnight incubation, we will extract an equal volume of bacterial culture and inoculate it into a 96-well plate containing LB medium and sodium butyrate. The plate will then be incubated at 37°C with shaking in a microplate reader, simultaneously measuring fluorescence intensity and bacterial growth curve.

E. coli strains transfected with either A segment or B segment were cultivated in the presence of 10mM and 20mM sodium butyrate. The results revealed that the sensitivity of A segment to sodium butyrate and its ability to enhance the expression of downstream genes were superior to those of B segment. In the end, we opted for the design of the A segment.

Reference

[1]. Schwan W. R. (2011). Regulation of fim genes in uropathogenic Escherichia coli. World journal of clinical infectious diseases, 1(1), 17–25. https://doi.org/10.5495/wjcid.v1.i1.17.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 The Plee promotor is under the control of PchA, we designed and compared Ppcha-pcha-Plee-egfp and Ppcha-pcha-egfp circuit.
 ===Experiment results===
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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/part-plee2.jpg"width="466" height="240"></center></html>
+<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/part-plee2.jpg"width="300" height="150"></center></html>
 We designed two gene fragments as mentinoned: one directly attaching EGFP downstream of the pchA promoter, and the other appending EGFP after the existing gene segment in enterohemorrhagic E. coli. We will compare the responsiveness and feedback levels of these two fragments to butyrate.
 We cloned the synthesized A segment and B segment onto the pet-28a plasmid. Subsequently, we will individually transform these plasmids into Escherichia coli BL21. After plating and overnight incubation, we will extract an equal volume of bacterial culture and inoculate it into a 96-well plate containing LB medium and sodium butyrate. The plate will then be incubated at 37°C with shaking in a microplate reader, simultaneously measuring fluorescence intensity and bacterial growth curve.