Part:BBa_K5348020
pYC-pKC-pL-RBS3-mCherry
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 5847
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4560
Illegal NgoMIV site found at 4632
Illegal NgoMIV site found at 4722
Illegal NgoMIV site found at 4740
Illegal NgoMIV site found at 5232
Illegal NgoMIV site found at 5525
Illegal NgoMIV site found at 5619
Illegal AgeI site found at 4274
Illegal AgeI site found at 5400 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 5289
Illegal BsaI.rc site found at 4173
Illegal SapI site found at 1
Illegal SapI.rc site found at 3967
pYC-pKC-pL-RBS3-mCherry (BBa_K5348020)
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. Our experimental results demonstrate that we can regulate the intensity of the light control system through the RBS replacement strategy.
Construction Design
This composite part consists of the pL-RBS3-mCherry (BBa_K5348011) and pTrc99k-backbone (BBa_K3999002), which was constructed in the E. coli DH5α strain. 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 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(3), and RBS(3)-mCherry fragments, and then ligated the pL-2-RBS(3) and RBS(3)-mCherry fragments by overlapping PCR to obtain the pL-2-RBS(3)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(3)-mCherry fragments, and the pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(3)-mCherry plasmid (Figure 2B).
Measurement: Light Control Test
Subsequently, we conducted light-control tests on the strain containing the pYC-pKC-pL-RBS(3)-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, no fluorescence values were detected for pL-RBS(3)-mCherry when cultured in both dark and blue light conditions, indicating that the strength of this RBS was too low (decreased by 150-fold) to initiate mCherry translation.
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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