Difference between revisions of "Part:BBa K5083099"

Latest revision as of 11:49, 15 September 2024

pCspA-T4 holin-T4 Lysozyme

To prevent gene leakage, we designed an engineered bacterial suicide system. We used the low-temperature inducible promoter BBa_K4987003 as a switch to express lysozyme, selected BBa_B0034 as the ribosome binding site, and then connected the lysozyme coding sequences BBa_K5083004 and BBa_K5083005, with BBa_B0015 as the terminator. Low temperatures enhance the activity of the CspA promoter, increasing the transcription level of the CspA gene. Under cold shock conditions, CspA gene expression is mainly regulated post-transcriptionally, with its mRNA secondary structure showing different stability and translation efficiency at 37°C and low temperatures. Cold induction leads to a temperature-dependent rearrangement of CspA mRNA's secondary structure, with the "cold shock" structure formed at low temperatures having higher translation efficiency and lower degradation rates compared to the structure at 37°C. 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 with arabinose or at low temperatures. 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 cell wall. This disruption compromises the integrity of the cell wall, disturbs the osmotic balance within the cell, leads to rapid leakage of the cytoplasm, and ultimately results in bacterial cell lysis and death.

Usage and Biology

Our team used a low-temperature inducible promoter to express the lytic proteins T4 holin and T4 lysozyme, with BBa_B0015 used as the terminator. We recombined the fragments into the plasmid pET23b and transformed the constructed plasmid into E. coli DH5α.

Fig 1.gene circuit diagram

Potential application directions

Under the low-temperature condition of 16°C, the engineered bacteria containing the lysozyme sequence remain at a low density, with the OD600 almost constant at 0.3.

Figure 2. The low-temperature inducible promoter causes the engineered bacteria to undergo cell death.

Sequence and Features