Part:BBa_K5348014

pL-RBS2-MazF

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-RBS2-MazF (BBa_K5348014)

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 the light-controlled regulation of the toxin protein MazF as a model protein, and the experimental results showed that we successfully realized the regulation of bacterial growth through this composite part.

Construction Design

This composite part consists of the pL-RBS2 (BBa_K5348005) and toxin protein MazF (BBa_K1096002). With the pL light-control system, we hope to regulate MazF expression in the dark and under blue light as a way to control bacterial growth.

Figure 1. Schematic diagram of the pL-RBS2-MazF

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 amplified the pL-1, pL-2-RBS(2), RBS(2)-MazF, and pTrc99k backbone fragments, respectively. To improve the efficiency of homologous recombination, we first used overlap PCR to obtain the pL-2-RBS(2)-MazF fragment, and then we homologous recombined the pL-1, pL-2-RBS(2)-MazF, and pTrc99k backbone fragments. Colony PCR and sequencing results confirmed the successful construction of the pYC-pKC-pL-RBS(2)-MazF plasmid (Figure 2B).

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

Measurement: Light Control Test

We conducted light-control tests on the successfully constructed strains. Results showed that under blue light cultivation, pL-RBS(2)-MazF reduced bacterial concentration (OD600) by 1.6 times compared to dark conditions. This indicates that under blue light, the toxic protein MazF was successfully expressed and inhibited bacterial growth, demonstrating that the pL element can regulate MazF expression (Figure 3).

Figure 3. Light-control tests 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.