Designed by: Wei Chung Kong   Group: iGEM15_Oxford15   (2015-08-28)

pLsr + RBS + T4 Holin

This composite part consists of the AI-2 quorum sensor-inducible promoter BBa_K117002, a weak ribosome binding site BBa_B0031, and a T4 Holin coding sequence BBa_K112805.


1. Lsr Quorum Sensing

The Lsr quorum sensing pathway is a non-species specific cell-to-cell communication pathway used by both Gram-positive and Gram-negative bacteria and is found in at least 55 strains of them, most notably strains of E. coli, S. typhimurium, and V. harveyi [1][2]. Production of the chemical messenger AI-2 is mediated by the enzymes Pfs and LuxS, which metabolizes the toxic metabolic product S-adenosylhomocysteine (SAH) in a stepwise manner to form AI-2 [3].

The transporter protein cluster LsrACDB (depicted as the gray channel through which AI-2 passes to enter the host cell) facilitates the uptake of AI-2 [4]. LsrR represses the pLsr promoter, which endogeneously is upstream of the lsrACDBFG and lsrRK operons, but in the context of synthetic biology can be placed upstream of any coding sequence of interest. LsrK phosphorylates and hence activates AI-2, allowing it to bind to LsrR and relieve its repression on pLsr, thus inducing the expression of the gene downstream of pLsr via an indirect mechanism [4].

2. T4 Holin

Holins are proteins that cause the formation of lesions in the bacterial cell membrane, causing it to be permeabilized. They are used by bacteriophages in conjunction with endolysins, which degrade the bacterial peptidoglycan layer, to achieve a precisely-timed lysis of the host bacterial cell, which is important for their propagation as they want to trigger cell lysis at the appropriate cell density such that the chances of newly-liberated phage particles attaching to uninfected host cells are maximized [5].


By cloning this part along with BBa_K1659000 into a single bacterial strain, ideally a luxS deletion mutant of E. coli or another species with the appropriate lsr machinery, we will in effect create an AI-2-responsive holin-endolysin host cell lysis system. Artificial endolysin "Artilysin" Art-175 would be kept under chemical induction, and in the presence of AI-2 secreting pathogens holin production would be triggered, leading to host cell lysis as well as the release of a concentrated pulse of Art-175 into the extracellular vicinity. We are interested in testing the potential of this "pulse antibacterial" system against pathogens involved in urinary tract infections, such as P. aeruginosa and E. coli.


[1] Surette, M.G., Miller, M.B. & Bassler, B.L., 1999. Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production. Proceedings of the National Academy of Sciences of the United States of America, 96(4), pp.1639–1644.

[2] Bassler, B.L., Greenberg, E.P. & Stevens, A.M., 1997. Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. Journal of Bacteriology, 179(12), pp.4043–4045.

[3] Brito, P.H. et al., 2013. Natural genome diversity of AI-2 quorum sensing in Escherichia coli: Conserved signal production but labile signal reception. Genome Biology and Evolution, 5(1), pp.16–30.

[4] Li, J. et al., 2007. Quorum sensing in Escherichia coli is signaled by AI-2/LsrR: Effects on small RNA and biofilm architecture. Journal of Bacteriology, 189(16), pp.6011–6020.

[5] Ramanculov, E. & Young, R., 2001. Genetic analysis of the T4 holin: Timing and topology. Gene, 265(1-2), pp.25–36.