Part:BBa_K5127016

Arabinose inducible Lambda Red system with reporter

This is the first plasmid for genome integration utilizing the Lambda Red recombination system. The other two plasmids within genome integration(BBa_K5127015)(BBa_K5127007). In our platform design, Tes4 is intended to produce butyrate in response to the absence of a specific metabolite. We aim to model the expression of Tes4, which in turn leads to an increase in butyrate levels and its eventual impact on the colonization or growth of our probiotic strain. We want to achieve butyrate production without giving the bacteria too much burden, so we decided to try genome integration of the Tes4 enzyme. To achieve this, we utilized the Lambda Red recombination system to integrate Tes4 and a reporter gene, GFP, into the genome of E. coli MG1655. (Yu et al., 2008) This allows us to track the expression of Tes4 through GFP fluorescence and correlate it with butyrate production and its downstream effects on our probiotic growth and colonization.

Team:BNDS-China 2024

Design

The pRed-Aspink expresses the Lambda Red recombinase under the control of an arabinose-inducible promoter. The plasmid also constitutively expresses the chromoprotein, AsPink, which serves as a visual marker to confirm the presence of the plasmid in the host cells (Figure 1).

Figure 1. Plasmid design of pRed-Aspink. Created by biorender.com.

Results

The Lambda Red recombinase proteins was derived from BNDS-China previous plasmid stock, and Aspink protien was derived from 2023 iGEM distribution kit. We used Golden Gate Assembly to construct pRed-Aspink. PCR and Gel Electrophoresis were performed to verify the success in constructing the fragment and backbone of the full plasmid (Figure 2).

Figure 2. Figure 2. The Agarose gel electrophoresis result of the PCR products of pReplace construction. A,B, materials to construct pReplace. C, golden gate assembly result of pReplace construction. The band at 6910bp in (C) indicated the success in plasmid construction.

The plasmid was transformed into E. coli MG1655, with the colonies displaying a pink color, indicating the successful construction and transformation of the plasmid.

Reference

Byung Jo Yu, Kui Hyeon Kang, Jun Hyoung Lee, Bong Hyun Sung, Mi Sun Kim, & Sun Chang Kim. (2008). Rapid and efficient construction of markerless deletions in the Escherichia coli genome. Nucleic Acids Research, 36(14), e84–e84. https://doi.org/10.1093/nar/gkn359

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1941
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1776
  Illegal AgeI site found at 3428
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1758