Difference between revisions of "Part:BBa K5186020"

Revision as of 03:40, 3 December 2024

2Pa-T4L

The 2Pa-T4L, encoding for a fusion protein of two amphipathic Pa peptides and T4 phage lysozyme(T4L), a lytic protein possesses the ability to degrade peptidoglycan, effectively targeting E. coli, thereby causing the autolysis of E. coli. Each Pa peptide is fused to the N-terminus of T4L via a flexible GSA peptide linker(Sequence: GSAGSAAGSG). It is reported that T4L's lytic process can be enhanced by fusing cell-penetrating peptides. And the efficiency of lysis is significantly influenced by the sequence and quantity of the incorporated cell-penetrating peptides.

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.

Pa peptide is an amphipathic peptide. 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.

T4L's lytic process can be enhanced by fusing cell-penetrating peptides. And the efficiency of lysis is significantly influenced by the sequence and quantity of the incorporated cell-penetrating peptides. With this strategy, the Xia Wu team has remarkably enhanced the lytic process, ensuring that over 99.97% of cells are lysed within 30 minutes post-induction, regardless of their age. (Xia Wu et al, 2021)

By fusing two Pa peptides to the N-terminus of T4L via flexible GSA peptide linker(Sequence: GSAGSAAGSG), 2Pa-T4L is created and expected to outperform T4L and Pa-T4L in terms of lysis efficiency.

This engineered component, designed for inducible expression, allows for the precise control of cell lysis. Such control is not only vital for a kill switch mechanism but also for the rapid recovery of recombinant proteins and other cellular contents, without the need for mechanical or chemical cell disruption. It can simplify the purification process, reducing both operational complexity and the risk of product degradation. (Jian Zha et al., 2021; Cuiping Pang et al., 2022)

T4 bacteriophage

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.The data are the means ± SD of three parallel replicate experiments.

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