Regulatory

Part:BBa_K4656004

Designed by: Xinji Gu   Group: iGEM23_NMU-China   (2023-08-08)
Revision as of 13:12, 5 October 2023 by GuXinji (Talk | contribs) (→‎Experimental results)

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.

Experimental results





We designed two gene fragments as mentinoned: one directly attaching to 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 in a microplate reader shaken at 37°C, 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，which involves plee promotor.

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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