Part:BBa_K5348032

pLEVI2.0

LEVI2.0 composite part insert into pCDFDuet dual expression vector

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
  COMPATIBLE WITH RFC[1000]

Construction Design

This composite part consists of BBa_J23116 promotor, LEVI (BBa_K5348029), rrnB T1 terminator (BBa_B0015), CoIE promotor (BBa_K2244006), repressor cI (BBa_K327018), PR promotor (BBa_R0051), RBS(B0034), and pCDF-DUET-1-backbone (BBa_K5348031) 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 representation of the (A) LEVI component [1], and (B) LEVI-based light control system.

Experimental Approach

Using the pYC-pKC-pL plasmid as a template, we introduced BglII and EcoRV restriction sites upstream and downstream of the cI-rrnB_T1-PR fragment, obtaining a BglII-cI-rrnB_T1-PR-EcoRV fragment. Then, using this fragment and the synthesized plasmid pCDF-LEVIsemi-on as templates, we performed BglII and EcoRV double digestion. After obtaining the digested fragments and backbone, we used T4 ligase for ligation and transformed the ligation product into DH5α competent cells. Colony PCR and sequencing results confirmed the successful acquisition of the pLEVI2.0 plasmid (Figure 2).

Figure 2. Construction results of pLEVI2.0 plasmid. (A) Design of pLEVI2.0 plasmid construction. (B-C) Amplification results of fragments with arrows indicating the correct bands. (D) Colony PCR results. (E) Sequencing results.

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.