Part:BBa_K5127012
Genome Integration of butyrate production protein (pReplace-Tes4)
Usage and Biology
The pReplace construction is designed for the integration of GOI into the E.coli MG1655 genome, with GFP downstream serving as a reporter gene to visualize successful integration. The two red features in the figure represent LP-2 homologous regions, which are 500bp sites on the genome utilized for integrase λ-Red mediated plasmid integration. Additionally, stringent ORI: R6K is incorporated, requiring a specialized protein for replication that most bacteria do not produce. This ensures that upon entering the host cell, the plasmid either cannot replicate and is eliminated or becomes integrated into the chromosome and replicates alongside it. This approach guarantees successful plasmid integration into the genome.
Team: BNDS-China 2024
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.
Design
Our team has replaced the barcode sequence with Tes4 and subsequently linked it with GFP in MG1655. This modification aims to integrate Tes4 into the genome for further characterization and validation of its ability to effectively produce butyrate.
Construction
pReplace was designed to facilitate the integration of the Gene of Interest (GOI) into the genome. This plasmid includes a barcode for colony PCR verification post-integration(Figure 1).
Figure 1. Plasmid design of pReplace-Tes4. Created by biorender.com.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 501
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 129
Illegal NheI site found at 152
Illegal PstI site found at 501 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 530
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 501
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 501
Illegal AgeI site found at 923 - 1000COMPATIBLE WITH RFC[1000]
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