Part:BBa_K5083098
pBAD-T4 holin-T4 Lysozyme
To prevent gene leakage, we designed an engineered bacterial suicide system. We used the arabinose promoter BBa_I13453 as an inducible switch to express lysozyme, and selected BBa_B0034 as the ribosome binding site. We then connected the lysozyme coding sequences BBa_K5083004 and BBa_K5083005, and used BBa_B0015 as the terminator. In the absence of arabinose, the araC protein binds to pBAD, blocking subsequent translation. In the presence of arabinose, the araC protein undergoes a conformational change, preventing it from binding to pBAD and inducing lysozyme expression. Finally, we recombined the fragment into the plasmid pET23b and transformed the constructed plasmid into Escherichia coli DH5α.
Description
The T4 holin protein, derived from the T4 bacteriophage, has a transmembrane domain that damages the cell membrane, allowing the phage's peptidoglycan-degrading enzyme to enter the periplasmic space and induce cell lysis. We successfully expressed the T4 holin protein in Escherichia coli DH5α and achieved cell lysis under induction by arabinose or low temperature. T4 Lysozyme is a small, globular protein composed of 164 amino acids. It initially binds to the bacterial cell wall, where it recognizes and cleaves the chemical bonds between N-acetylglucosamine and N-acetylmuramic acid in the bacterial cell wall. This disruption compromises the integrity of the cell wall, disturbs the osmotic balance within the cell, leading to rapid leakage of the cytoplasm, and ultimately results in bacterial cell lysis and death.
Usage and Biology
Our team used the arabinose promoter to induce the expression of the T4 holin and T4 lysozyme lytic proteins, with BBa_B0015 as the terminator. We recombined this fragment into the plasmid pET23b and transformed the constructed plasmid into Escherichia coli DH5α.
Fig 1.Gene circuit diagram
Potential application directions
In the absence of arabinose, the araC protein binds to pBAD, blocking subsequent translation. In the presence of arabinose, araC undergoes a conformational change that prevents it from binding to pBAD, thereby inducing the expression of lysozyme. We verified the system's effectiveness by feeding arabinose to the culture medium. The results showed that under 0.5 mM/L arabinose induction, the OD600 of the culture containing the arabinose promoter suicide system was significantly lower than that of the control, demonstrating the effectiveness of the suicide system (Fig 2).
Fig 2.pBAD induces lysozyme expression
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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