Part:BBa_K5348008
pL-RBS0-mCherry
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1882
- 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.rc site found at 200
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BBa_K5348008 (pL-RBS0-mCherry)
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, and tested its light-controlled regulatory function using mCherry as a model protein. The following test results of the original RBS show that the element can regulate mCherry expression, but there is leakage.
Construction Design
This composite part consists of the BBa_K3447133 (hereafter referred to as the pL) and fluorescent protein mCherry (BBa_K3822002). With the pL light-control system, regulation of mCherry expression in the dark and under blue light can be achieved.
Engineering Principle
The pL light-control system consists of several basic parts. Under dark conditions, 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(0) and RBS(0)-mCherry fragments, and then ligated the pL-2-RBS(0) and RBS(0)-mCherry fragments by overlapping PCR to obtain pL-2-RBS(0)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(0)-mCherry fragments, and pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(0)-mCherry plasmid (Figure 2B).
Measurement: Light Control Test
Subsequently, we conducted light-control tests on the strain containing pYC-pKC-pL-RBS(0)-mCherry plasmid. We cultured the strains under dark conditions 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. However, it was observed that the pL light-control system exhibited leakage, with detectable increases in mCherry RFU after culturing for more than 8 hours under dark conditions.
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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