Difference between revisions of "Part:BBa K5348005"

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pL-RBS2

Sequence and Features

BBa_K5348005 (pL-RBS2)

Summary

This is an improved new part based on existing part: BBa_K3447133 (light-on induced system). To reduce the leaky expression of this existing part: BBa_K3447133, we reduced the strength of the RBS, which is connected to the target genes. We tested the fluorescent protein mCherry and the toxin protein MazF as model proteins, respectively. The experimental results showed that we succeeded in reducing the leakage of the system and achieved the regulation of bacterial growth.

Construction Design

To reduce the leaky expression of the light-inducible induction system (BBa_K3447133, hereafter referred to as the pL-RBS0), we reduced the intensity of the RBS linked to the target gene in this element by 108-fold (RBS2). The composition of this element is shown below.

Figure 1. Schematic diagram of the pL-RBS2.

Engineering Principle

Under dark condition, histidine kinase (YF1) phosphorylates FixJ (response regulator of histidine kinase), which activates PFixK2 (the target gene for transcription upon FixJ activation), driving the expression of the cI gene (λ phage repressor), which represses the transcription of its cognate promoter, PR (the cognate promoter of cI), and downstream genes cannot be expressed. Under blue light, the cI gene cannot be expressed, PR can be transcribed normally, and downstream genes can be expressed [1].

Experimental Approach

The plasmid construction scheme is shown in Figure 2A. We synthesized the pL element at GenScript and divided it into two fragments, pL-1 and pL-2, for synthesis. We amplified pL-1, pL-2-RBS(2) and RBS(2)-mCherry/MazF fragments, and then ligated the pL-2-RBS(2) and RBS(2)-mCherry/MazF fragments by overlapping PCR to obtain pL-2-RBS(2)-mCherry/MazF fragments. Finally, we ligated pL-1, pL-2-RBS(2)-mCherry/MazF fragments, and pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(2)-mCherry/MazF plasmids (Figure 2B-C).

Figure 2. Construction results of pYC-pKC-pL-RBS(2)-mCherry/MazF plasmids. (A) Construction Strategy. (B-C) Colony PCR and sequencing results of pYC-pKC-pL-RBS(2)-mCherry/MazF.

Measurement: Light Control Test

Subsequently, we conducted light-control tests on the strain containing pYC-pKC-pL-RBS(2)-mCherry plasmid. We cultured the strains under dark condition and blue light irradiation, respectively, sampling at intervals to measure the RFU (relative fluorescence units) of the bacterial suspension. As shown in Figure 3, the test results verified that the pL light-control element could regulate mCherry expression under dark and blue light conditions. As the RBS strength decreased, the RFU of mCherry decreased accordingly, indicating that the RBS replacement strategy can achieve regulation of the pL light-control system.

Figure 3. Light-control test results of pL-RBS(2)-mCherry.

Finally, we conducted light-control tests on the strain containing pYC-pKC-pL-RBS(2)-MazF plasmid. Results showed that under blue light cultivation, pL-RBS(2)-MazF reduced bacterial concentration (OD600) by 1.6 times compared to dark conditions. This indicates that under blue light, the toxic protein MazF was successfully expressed and inhibited bacterial growth, demonstrating that the pL element can regulate MazF expression. (Figure 4).

Figure 4 Light-control tests results pL-RBS(2)-MazF.

Reference

[1] H, Mays RL, Hoffman SM, Avalos JL. Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS Synth Biol. 2021 Aug 20;10(8):2015-2029.