Part:BBa_K5348035

pLEVI2.0-CcdB

pLEVI2.0-CcdB

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1332
  Illegal NheI site found at 3548
  Illegal NheI site found at 3571
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 71
  Illegal XhoI site found at 120
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 90
  Illegal NgoMIV site found at 585
  Illegal AgeI site found at 332
  Illegal AgeI site found at 4253
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5729
  

Construction Design

This composite part consists of pLEVI2.0 (BBa_K5348032) and toxin protein CcdB (BBa_K3512001), which was constructed in E. coli DH5α strain.

Engineering Principle

Under blue light irradiation, the LEVI domain undergoes a conformational change, forming a protein dimer. This dimer binds to its homologous operator sequence, inhibiting PColE promoter activity. Consequently, the cI repressor cannot be expressed, allowing the target protein to be expressed. In dark conditions, the PColE promoter initiates transcription and expression of the cI repressor, thereby inhibiting target gene transcription [1]. Additionally, we used the CloDF13 ori, a low-copy replication origin more suitable for constructing plasmids containing toxic genes. Our modified light-control system, LEVI2.0, is shown in Figure 1.

Figure 1. Schematic diagram of pLEVI2.0-CcdB.

Experimental Approach

Using pYC-pKC-pL-CcdB plasmid as a template, we introduced AatII and XhoI restriction sites upstream and downstream of the CcdB fragment, obtaining the AatII-CcdB-XhoI fragment. We then performed double digestion (AatII and XhoI) on both the AatII-CcdB-XhoI fragment and pLEVI2.0 plasmid to obtain the digested fragment and backbone (Figure 2).

Figure 2. Construction results of the pLEVI2.0-CcdB plasmid. (A) Design of pLEVI2.0-CcdB plasmid construction. (B) Amplification results of the CcdB fragment. (C) Enzyme digestion of the vector.

Due to time constraints, we have not been able to complete the linkage of the fragments to the backbone, transformation, and light-control tests. Our expected result is that the strain does not express or expresses a small amount of CcdB when cultured under dark conditions. In contrast, under high-intensity blue light, the strain can express CcdB protein, resulting in impaired strain growth.

References

[1] Chen, X., Liu, R., Ma, Z. et al. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Res. 2016, 26, 854–857.