Part:BBa_K1835500
Phi X 147 E Lysis Gene
The phi X 174 bacteriophage is a phage that infects E. coli. The You Lab here at Duke shared with us a strain of bacteria carrying a plasmid which contained the phi X 174 bacteriophage’s E protein, which induces lysis in bacteria. Lysis is caused by a transmembrane tunnel created by the E protein. It is believed that the transmembrane tunnel forms as a result of the E protein inhibiting peptidoglycan synthesis during cell growth.
One example of an application for this cell death gene is that employed by Lingchong You, et al. They designed a construct that contained the lysis gene as well as quorum sensing components, which caused populations oscillations over time. The gene circuit did not work as anticipated, but the oscillations were a product of cell suicide caused by the E lysis gene.
The figure below graphically displays the E lysis protein’s effect on cell growth. The uninduced sample grew approximately four times as well as the fully induced sample.
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Characterization
After assembling these autolytic genes to IPTG-inducible expression cassette in E. coli (Figure 1a), strains KS 1-4 were successfully constructed as it is shown in figure 1b. Based on the growth curve analysis, strains KS1-4 showed growth reduction post-induction and different kill switch designs have varying effectiveness in killing E. coli (Figure 1c).
Reports suggest that T4L's lytic efficiency can be enhanced by fusing it with cell-penetrating peptides like Pa, yet the growth curves of strains KS 2, with an additional Pa peptide, were identical. Strain KS 1 with two Pa peptides, showed E. coli regaining growth, contradicting the report.
Ultimately, our tests show that ΦX174-E expression cassette (KS 4) outperforms T4L, Pa-T4L, and 2Pa-T4L, with the majority of cell lysis within one hour of induction. This makes gene E an optimal candidate for our kill switch setting.
Figure 1. Various autolytic genes expression cassettes are engineered for kill switch setting in E. coli strain DH5a. (a) Genetic circuit construction of strains KS 1-4. (b) Gel electrophoresis analysis of transformed autolytic genes expression cassettes. (c) Growth curve of control (E. coli strain DH5a) and strains KS 1-4.
Reference
Jian Zha, Zhiqiang Liu, Runcog Sun, Jonathan S. D., Xia Wu. Endolysin-Based Autolytic E.coli System for Facile Recovery of Recombinant Proteins. J. Agric Food Chem. 2021, 69. 3134-3143. https://doi.org/10.1021/acs.jafc.1c00059 <ds> Cuiping Pang, Song Liu, Guoqiang Zhang, Jingwen Zhou, Guocheng Du, Jianghua Li. Enhancing extracellular production of lipoxygenase in Escherichia coli by signal peptides and autolysis system. Microb Cell Fact. 2022, 21(1): 42. https://doi.org/10.1186/s12934-022-01772-x
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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