Part:BBa_K5348021

pYC-pKC-pL-RBS2-CcdB

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 5847
  
• 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 site found at 7301
  Illegal BsaI.rc site found at 4173
  Illegal SapI site found at 1
  Illegal SapI.rc site found at 3967
  

pYC-pKC-pL-RBS2-CcdB (BBa_K5348021)

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), toxin protein CcdB (BBa_K3512001), and pTrc99k-backbone (BBa_K3999002). 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.

Figure 1. Schematic diagram of pYC-pKC-pL-RBS2-CcdB

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).

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

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.

Figure 3. Sequencing results of pYC-pKC-pL-RBS(2)-CcdB plasmid in DH5α strain.

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.