Part:BBa_K5348012
pL-RBS2-CcdB
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1874
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 587
Illegal NgoMIV site found at 659
Illegal NgoMIV site found at 749
Illegal NgoMIV site found at 767
Illegal NgoMIV site found at 1259
Illegal NgoMIV site found at 1552
Illegal NgoMIV site found at 1646
Illegal AgeI site found at 301
Illegal AgeI site found at 1427 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1316
Illegal BsaI site found at 3328
Illegal BsaI.rc site found at 200
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pL-RBS2-CcdB (BBa_K5348012)
Summary
To reduce the leaky expression of the light-on induced system (BBa_K3447133), we reduced the strength of the RBS, which is connected to the target genes. We tested its light-controlled regulatory function using the toxin protein CcdB as a model protein.
Construction Design
This composite part consists of the pL-RBS2 (BBa_K5348005) and toxin protein CcdB (BBa_K3512001). With the pL light-control system, we hope to regulate CcdB expression in the dark and under blue light as a way to control bacterial growth.
Engineering Principle
The pL light-control system consists of several basic parts. 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)-CcdB fragments, and then ligated the pL-2-RBS(2) and RBS(2)-CcdB fragments by overlapping PCR to obtain the pL-2-RBS(2)-CcdB fragment. Finally, we ligated pL-1, pL-2-RBS(2)-CcdB fragments, and the pTrc99k vector by Gibson assembly. Given the high toxicity of the CcdB protein, we first constructed it in the CcdB-resistant E. coli DB3.1 strain to obtain the plasmid. Colony PCR and sequencing results confirmed the successful construction of the pYC-pKC-pL-RBS(2)-CcdB plasmid (Figure 2B).
Subsequent Steps
Subsequently, we transformed the sequence-verified pYC-pKC-pL-RBS(2)-CcdB into E. coli DH5α competent cells to test their light-control effects. However, sequencing results for pYC-pKC-pL-RBS(2)-CcdB revealed fragment deletions after transformation into DH5α, indicating construction failure (Figure 3). This may be due to the high toxicity of CcdB to DH5α coupled with the leakage of the pL light control system, which exerted growth pressure on the strain even though the RBS intensity was 108-fold lower, resulting in the deletion of the introduced exogenous gene.
References
[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.
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