Part:BBa_K5348027

pHT43-pL-RBS2-mcherry

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1091
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3022
  Illegal BglII site found at 6103
  Illegal BamHI site found at 2905
  Illegal BamHI site found at 9975
  Illegal XhoI site found at 6107
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 8688
  Illegal NgoMIV site found at 8760
  Illegal NgoMIV site found at 8850
  Illegal NgoMIV site found at 8868
  Illegal NgoMIV site found at 9360
  Illegal NgoMIV site found at 9653
  Illegal NgoMIV site found at 9747
  Illegal AgeI site found at 2932
  Illegal AgeI site found at 8402
  Illegal AgeI site found at 9528
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 9417
  Illegal BsaI.rc site found at 8301
  

pHT43-pL-RBS2-mCherry (BBa_K5348027)

Construction Design

This composite part consists of the pL-RBS2-mCherry (BBa_K5348010) and pHT43-backbone (BBa_K3992003), which was constructed in the *Bacillus subtilis* WB800N strain.

Engineering Principle

The pL light-control system consists of several basic parts. Under dark conditions, 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 pHT43-pL-RBS2-mCherry

Experimental Approach

The plasmid construction scheme is shown in Figure 2A. First, using pYC-pKC-pL-RBS(2)-mCherry as a template, we amplified the pL-RBS(2)-mCherry fragment. Simultaneously, we obtained the pHT43 vector fragment through inverse PCR. Then, we connected the fragment with the vector through homologous recombination and transformed them into DH5α competent cells. Colony PCR and sequencing results confirmed the successful acquisition of the plasmid (Figure 2B).

Figure 2. Construction results of pHT43-pL-RBS(2)-mCherry plasmid. (A) Construction Strategy. (B) Colony PCR and sequencing results.

Next, we transferred the successfully constructed plasmid into *B. subtilis* WB800N by electrical transformation. We performed colony PCR identification and sequencing on the resulting transformants, confirming the successful acquisition of *B. subtilis* strain containing pHT43-pL-RBS(2)-mCherry (Figure 3).

Figure 3. (A) Positive transformants and (B) sequencing results of the strain.

Measurement: Light Control Test

Finally, we conducted light-control tests on these strains. Frustratingly, after incubating *B. subtilis* strains containing pHT43-pL-RBS(0/1/2/3)-mCherry for 48 h in darkness and blue light, respectively, the organisms showed almost no visible change in color (Figure 4).

Figure 4. Light-control tests on strains containing the pHT43-pL-RBS(n)-mCherry plasmids.

Challenges and Future Plans

After literature research, we found that *B. subtilis* often faces challenges in heterologous protein expression. Its protein hydrolysis mechanism degrades improperly folded or excessively folded proteins, resulting in low yields. Codon usage bias between heterologous genes and the host translation machinery can also hinder expression efficiency. Therefore, optimization of the expression system, including codon optimization and the use of signal peptides, is often required to increase the yield of heterologous proteins in *B. subtilis* [2].

In our future plans, we intend to improve the system by:

1. Adding a signal peptide before the target protein to improve protein secretion.
2. Optimizing the codons of the target protein sequence to make it more suitable for the *B. subtilis* host.

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.

[2] Hwang, S. H., et al. (2017). "Challenges and strategies in *Bacillus subtilis* for heterologous protein expression." Biotechnology Advances, 35(3), 291-308.