Part:BBa_K5348011

pL-RBS3-mcherry

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1874
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1316
  Illegal BsaI.rc site found at 200
  

pL-RBS3-mCherry (BBa_K5348011)

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 BBa_K3447133 (hereafter referred to as the pL) with RBS mutants (BBa_K5348003) 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.

Figure 1. Schematic diagram of the pL-RBS3-mCherry

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).

Figure 2. Construction results of pYC-pKC-pL-RBS(3)-mCherry plasmid. (A) Construction Strategy. (B) Colony PCR and sequencing results.

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.

Figure 3. Light-control test results.

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.