Part:BBa_K5348016

pYC-pKC-pL

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 5855
  
• 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.rc site found at 4173
  Illegal SapI site found at 1
  Illegal SapI.rc site found at 3967
  

pYC-pKC-pL (BBa_K5348016)

Construction Design

This composite part consists of the light-on induced system (BBa_K3447133) and the pTrc99k-backbone (BBa_K3999002), which was constructed in the E. coli DH5α strain.

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].

Figure 1. Schematic diagram of the pYC-pKC-pL

Experimental Approach

The light-control element pL (BBa_K3447133) was derived from literature and validated by iGEM20_Jilin_China [2]. We synthesized the pL element at GenScript company, which was divided into two fragments for synthesis, pL-1 and pL-2. These fragments were then ligated to the pTrc99k vector by Gibson assembly (Figure 2A). As shown in Figure 2B-D, we successfully amplified pL-1, pL-2, and the pTrc99k vector fragment. After DNA gel extraction, we used a multi-fragment homologous recombination kit to recombine these three fragments and then transformed them into DH5α competent cells. Colony PCR and sequencing results confirmed the successful construction of this plasmid.

Figure 2. Construction scheme and results of pYC-pKC-pL plasmid. (A) Design of pYC-pKC-pL plasmid construction. (B) Amplification results of fragments with arrows indicating the correct bands. (C) Medium plates incubated overnight after transformation. (D) Sequencing results of plasmid.

Measurement: Light Control Test

We conducted light-controlled growth tests on strains containing the pYC-pKC-pL plasmid to determine whether the pL element itself would affect bacterial growth. We cultured the strain under both dark conditions and blue light irradiation, sampling at intervals to measure the OD600 of the bacterial suspension. The light-control results showed no difference in growth between blue light and dark conditions for strains containing only the pL light-control element, indicating that the element itself does not affect strain growth (Figure 3).

Figure 3. Light-controlled growth tests on strains containing the pYC-pKC-pL 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.