Part:BBa_K5186018
T4L
Description
The T4L, encoding for the T4 phage lysozyme(T4L), possesses the ability to degrade peptidoglycan, effectively targeting E. coli, thereby causing the autolysis of E. coli.
This is a part of a part collection where we enable the autolysis of E.coli. To find out the most efficient autolysis gene, T4L(BBa_K5186018), Pa-T4L(BBa_K5186019), 2Pa-T4L(BBa_K5186020), X174E(BBa_K1835500) are engineered to be IPTG-inducibly expressed and tested, and thus make up a part collection. This collection serves as a valuable resource for the iGEM community and researchers for kill switch settings in E. coli and recovery of recombinant proteins or other intracellular products without the need for mechanical or chemical cell disruption methods.
Usage and biology
T4L is a coding sequence that encodes the T4 lysozyme enzyme, a bacteriophage-origin protein with potent lytic capabilities against Escherichia coli. It works by degrading the peptidoglycan layer of the bacterial cell wall, which is crucial for maintaining cell shape and integrity. Once the peptidoglycan is broken down, the cell wall loses its strength, leading to cell lysis.
When this part is designed to be inducibly expressed, the controlled expression enables the rapid lysis of E. coli cells for the kill switch setting or recovery of recombinant proteins or other intracellular products without the need for mechanical or chemical cell disruption methods. (Jian Zha et al., 2021; Cuiping Pang et al., 2022)
Source
T4 bacteriophage.
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
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]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 255
Illegal AgeI site found at 325 - 1000COMPATIBLE WITH RFC[1000]
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