Difference between revisions of "Part:BBa K5127015"

Revision as of 02:15, 2 October 2024

Characterization device for P2 promoter in B. subtilis

This is the second plasmid for genome integration utilizing the Lambda red recombination system. The other two plasmids within genome integration(BBa_K5127016)(BBa_K5127007).

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

The second plasmid for genome integration, pDual-Select, was designed to facilitate both positive and negative selection. Initially, chloramphenicol resistance is used for positive selection, enabling the identification of colonies with successful transformation and recombination of pDual-Select onto the genome. Following recombination by the Lambda Red system, the expression of SacB protein allows for negative selection, as SacB is lethal in the presence of sucrose. This step selects for colonies with the correct integration of the gene of interest replacing the CmR-SacB expression cassette.

The pDual-select plasmid was assembled with an R6K origin of replication (ori), which can only replicate in E. coli DH5-alpha pir+ strain. This design ensures that when the plasmid is transformed into E. coli MG1655, they cannot replicate, preventing the presence of redundant plasmid copies and avoiding false-positive results during integration verification. In addition, the plasmid contains homology arms to guide presice integration of the GOI into the specific, non-essential regions of the E. coli MG1655's genome. This ensures that the integration occurs at safe sites, avoiding disruption of the organisms' basic functions and maintaining normal cellular processes.

Figure 1. Plasmid design of pDual-Select. Created by biorender.com.

Build

The sequence of homology arms and R6K Ori were obtained from BNDS-China's previous plamids. We used Golden Gate Assembly to construct pReplace. PCR and Gel Electrophoresis were performed to verify the success in constructing the fragment and backbone of the full plasmid (Figure 2).

Figure 2. The Agarose gel electrophoresis result of the PCR products of pReplace construction. A,materials to construct pReplace. B, golden gate assembly result of pReplace construction. The band at 5831bp in (B) indicated the success in plasmid construction.

Results

The plasmid was transformed into E. Coli Trelief 5-alpha for contruction, by plating them on the Kanamycin plates, the construction of this plasmid was being confirmed.

At the same time, the phase I cells were prepared as chemically competent cells and induced with arabinose for a 3-hour resuscitation. The pDual-Select plasmid was then transformed into these Phase I competent cells, which were subsequently plated on chloramphenicol plates for selection. Only bacteria with successful genome integration of the plasmid will grow on the plate, due to the lost of the second plasmid. (Figure 3.)

Figure 3. The plating result of phase II cells.

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