Part:BBa_K5083004
T4 holin
T4 Holin is a small membrane protein from the T4 bacteriophage. The Holin protein has two transmembrane domains, with the N-terminal and C-terminal located in the cytoplasm and the extracellular space, respectively. It features a relatively large cytoplasmic domain (about 32 residues) and a periplasmic domain (about 150 residues), and has only one transmembrane domain. The aggregation of these transmembrane domains creates damage in the membrane, allowing the bacteriophage's peptidoglycan-degrading enzymes to enter the periplasmic space, which then triggers cell lysis.
Description
Considering the potential risk of gene leakage from engineered strains, we have designed a suicide system using the T4 Holin protein from the T4 bacteriophage. The transmembrane domain of Holin can damage the cell membrane, allowing the bacteriophage's peptidoglycan-degrading enzymes to enter the periplasmic space, which subsequently triggers 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.
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 Holin. We then connected the coding sequence of T4 Holin and used the terminator (BBa_B0015) to complete the construct. The entire fragment was recombined into the plasmid pET23b, and the constructed plasmid was transformed into Escherichia coli DH5α.
Fig 1.gene circuit diagram
Potential application directions
Firstly, we successfully amplified the T4 Holin protein (Fig. 2). We then tested the survival status of Escherichia coli DH5α strains under different inducer conditions for T4 Holin expression. The results showed that in the presence of 0.5 mM arabinose, the OD600 of the bacterial culture began to significantly decline after 5 hours of incubation, reaching nearly zero by 20 hours (Fig. 3.A). In contrast, under the low-temperature condition of 16°C, the engineered bacteria containing the lysis enzyme sequence remained at a low density, with the OD600 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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