Part:BBa_K5083005

T4 endolysin

T4 Lysozyme is a small globular protein composed of 164 amino acids (Fig. 14, right). It first binds to the bacterial cell wall. T4 Lysozyme recognizes and cleaves the chemical bonds between N-acetylglucosamine and N-acetylmuramic acid in the bacterial cell wall, disrupting the integrity of the cell wall and disturbing the osmotic balance within the cell. This leads to rapid leakage of the cell contents, ultimately resulting in cell lysis and death of the bacterial cell [1].

Description

Considering the potential risk of gene leakage from engineered strains, we have designed a suicide system using the T4 Lysozyme protein from the T4 bacteriophage. T4 Lysozyme recognizes and cleaves the chemical bonds between N-acetylglucosamine and N-acetylmuramic acid in the bacterial cell wall. This disruption of the cell wall's integrity causes an imbalance in the internal osmotic pressure, leading to rapid leakage of the cell contents and ultimately resulting in cell lysis and death.

Usage and Biology

We used the arabinose operon (BBa_I13453) and the cold-inducible promoter pCspA (BBa_K4987003) as induction switches for the expression of T4 Lysozyme. The coding sequence of T4 Lysozyme was then connected and followed by the terminator (BBa_B0015). 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

Firstly, we successfully amplified the T4 Holin protein (Fig. 2). We then assessed the survival status of Escherichia coli DH5α strains expressing T4 Lysozyme under different induction conditions. The results showed that in the presence of 0.5 mM arabinose, the OD600 of the bacterial culture began to significantly decrease after 5 hours of incubation, approaching zero by 20 hours (Fig. 3.A). In contrast, under the low-temperature condition of 16°C, the engineered bacteria containing the lysozyme sequence maintained a low density, with the OD600 remaining almost constant at 0.3 (Fig. 3.B).

Figure 2. Verification of the target fragment by agarose gel electrophoresis

Figure 3. Induction of the suicide gene expression under different conditions(A) Arabinose-induced suicide gene expression(B) Low-temperature-induced suicide gene expression

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]