Part:BBa_K5348022

pYC-pKC-pL-RBS3-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-RBS3-CcdB (BBa_K5348022)

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 BBa_K5348006 (pL-RBS3), 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-RBS3-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(3), and RBS(3)-CcdB fragments, and then ligated the pL-2-RBS(3) and RBS(3)-CcdB fragments by overlapping PCR to obtain the pL-2-RBS(3)-CcdB fragment. Finally, we ligated pL-1, pL-2-RBS(3)-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 pYC-pKC-pL-RBS(3)-CcdB plasmid (Figure 2B).

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

Subsequent Steps

Subsequently, we transformed the sequence-verified pYC-pKC-pL-RBS(3)-CcdB into E. coli DH5α competent cells to test their light-control effects. Sequencing results showed that we successfully transferred pYC-pKC-pL-RBS(3)-CcdB into DH5α competent cells.

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

Measurement: Light Control Test

Finally, we conducted light-controlled growth tests on strains containing pYC-pKC-pL-RBS(3)-CcdB. Test results showed that pL-RBS(3)-CcdB cultured under dark conditions and blue light had little difference in bacterial concentration (OD600), and the OD600 was consistent with the negative control (Figure 4). These results indicate that the strength of RBS3 is too low to achieve regulation of the toxic protein CcdB.

Figure 4. Light-control tests on strains containing the pYC-pKC-pL-RBS(3)-CcdB plasmid.

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.