Part:BBa_K5322000
Constitutive Mfp3 Expression System
Nitric-oxide-inducible lysis module
Contents
Usage and Biology
Plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-PhiX174E-T7 leverages the pET29a vector backbone for high-level expression in Escherichia coli. It includes an engineered regulatory system composed of the strong constitutive promoter J23119 followed by the nitric oxide-inducible SoxR/SoxS system, controlling the expression of the lysis protein PhiX174E. This design allows the lysis protein to be expressed in response to elevated nitric oxide levels, a common indicator of inflammatory conditions in cellular environments. The ribosome binding site (RBS) ensures efficient translation of the mRNA, and the T7 terminator provides a clean and efficient end to transcription. This system is intended for use in synthetic biology applications where controlled cell lysis is necessary, such as in timed release of therapeutic compounds or biocontainment strategies.
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-Mfp3-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.
Figure 2-1 Plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-Mfp3-T7
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 572 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1009
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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