Part:BBa_K5322000

Constitutive Mfp3 Expression System

Nitric-oxide-inducible lysis module

Construction of the plasmid

In order to allow the strain to lysis at the appropriate time and release product extracellularly, we designed a lysis module based on the lysis protein PhiX174E (91aa). The lysis protein PhiX174E (91aa) is a protein encoded by the PhiX174 phage gene E. Studies have shown that the PhiX174E protein triggers cell lysis through membrane binding and oligomerization with the host cell, as well as proton-dependent steps. As shown in Figure 2-1, we designed the plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-PhiX174E-T7. Through homologous recombination integration, this plasmid was transferred to DH5α, and plasmid extraction was performed after picking E. coli single colonies on several transformation plates. We conducted PCR validation with specific primers, targeting a 1320bp fragment, as shown in Figure 2-2. Plasmids with correctly positioned bands were sent to GENEWIZ Co. for sequencing. The sequencing results in Figure 2-3 confirmed that the plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-PhiX174E-T7 was successfully constructed.

Figure 2-1 Plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-PhiX174E-T7

Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-PhiX174E-T7(1320bp)

Figure 2-3 plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-PhiX174E-T7 sequencing result

Protein Expression Validation

We verified the performance of the lysis module through dry experiments and plan to complete wet experimental validation of its lytic function in the future. Through literature review and numerical modeling validation, we found that after inducing the expression of antimicrobial peptides and lysis proteins with NO for twenty minutes, the engineered strain will be lysed by the lysis protein and release the antimicrobial peptides. Mathematical modeling confirmed that at this time, the concentration of antimicrobial peptides is sufficient to reach an effective inhibitory concentration.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 572
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1009
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]