Difference between revisions of "Part:BBa K5186021"

Latest revision as of 03:42, 3 December 2024

Ptac-RiboJ-RBS-X174E-B0015

Description

Ptac-RiboJ-RBS-X174E-B0015 is an expression cassette in E. coli expressing gene E(BBa_K1835500) from the ΦX174 bacteriophage, which encodes a transmembrane pore-forming protein that can cleave the host cell membrane to induce the leakage of intracellular proteins. With this expression cassette, gene E can be IPTG-inducibly expressed under the control of pTac-RiboJ (BBa_K3552015) in all strains 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.

The ΦX174 bacteriophage's Gene E encodes a transmembrane pore-forming protein that disrupts the host cell membrane, causing the release of intracellular proteins. This protein interacts with E. coli's cell wall synthesis proteins, forming a transmembrane structure. The expression of the phage lysis protein can either weaken or entirely lyse the cell membrane, resulting in E. coli cell death and additional protein leakage.

In our 2024 initiative to create a kill switch for E. coli, we have successfully incorporated the gene E expression cassette into E. coli DH5a. Our tests show that it 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 component in the parts collection. (Cuiping P. et al, 2020)

X174E is from ΦX174 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

Sequence and Features