Part:BBa_K5348017

pYC-pKC-pL-RBS0-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 5855
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE 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-RBS0-mCherry (BBa_K5348017)

Summary

To reduce the leaky expression of the light-on induced system (BBa_K3447133, hereafter referred to as the pL-RBS0), 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 pL-RBS0-mCherry (BBa_K5348008) and pTrc99k-backbone (BBa_K3999002), which was constructed in E. coli DH5α strain. 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 pYC-pKC-pL-RBS0-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 P_FixK2 (the target gene for transcription upon FixJ activation), driving the expression of cI gene (λ phage repressor), which represses the transcription of its cognate promoter, P_R (the cognate promoter of cI), and downstream genes cannot be expressed. Under blue light, the cI gene cannot be expressed, P_R 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 the pL-2-RBS(0)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(0)-mCherry fragments, and the pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(0)-mCherry plasmid (Figure 2B).

Figure 2. Construction results of pYC-pKC-pL-RBS(0)-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(0)-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. 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.

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.