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The Coliguard - Killing


After the detection of a contaminant by our ColiGuard System, part of the labor population must differentiate into the killer population, which has as its most remarkable feature the ability to kill this contaminant. Since our project aims at solving the contamination problem in the industrial production of ethanol, we developed a killing mechanism able to destroy the most important bacterial contaminant of the ethanol production process: the Lactobacilli, which is a group of Gram positive bacteria. But how can a bacterium kill another bacterium without killing itself? That was the first challenge to be solved in the development of this mechanism. As a possible answer we decided to focus on the main biological difference between our guard bacteria, E. coli, and the contaminant, Lactobacilli, which is Gram staining. While E. coli is a Gram negative species Lactobacilli belong to the group of Gram positive bacteria. Physiologically, Gram coloration translates into characteristics of the membrane and outer cell wall, with Gram negative showing two cell membranes surrounding a thin cell wall, which remains isolated from the intracellular and extracellular environment. On the other hand, Gram positive bacteria have a thick cell wall which surrounds the cell membrane and is in direct contact with the extracellular environment. With these differences in mind we propose a way to attack the exposed cell wall of Lactobacilli by secreting in the medium a substance capable of degrading the cell wall of Gram positive bacteria, while preserving the structure of the Gram negative E. coli. As a result, we chose lysozyme as our weapon, which is the most effective enzyme in the degradation of Gram positive cell walls. Once our weapon was chosen the second problem to be solved was how to engineer E. coli to secrete it? The Alpha Hemolysin Secretion System As a Gram negative bacterium, E. coli doesn’t have a well developed secretion system that allows the transport of proteins to the extracellular medium. However, the hemolysin alpha secretion system is the best one described so far. The alpha hemolysin secretion system is encoded by an operon containing four genes (Fig. 1A): hlyD and HlyB code for proteins that assemble into the transporter, HlyA codes for the hemolysin itself, and HlyC codes for a protein required for HlyA activation. In order to adapt the alpha hemolysin system to export our lysozyme, we intend to construct a device in Biobrick format containing HlyB, HlyD and 252 bp of the carboxy terminal region of the gene HlyA using the Silver Standard (Fig. 1B). These 252 bp code for the signal peptide that enable genes fused to it to be recognized by hlyB and hlyD and be transported outside the cell. This will be the first biobrick designed to make E. coli secrete a protein using a transport system and can be applied to a big range of targets, thus helping solve the secretion problem of E. coli. We then intend to fuse to this device a part coding for the lambda phage’s lysozyme without the stop codon (Fig. 2). As a result, we hope that our lysozyme with the signal peptide will be secreted outside the cell and thus be able to kill and destroy Lactobacilli and other Gram positive contaminants who dare to stay on our way.

Since it has been reported that this secretion system doesn’t work for all proteins, the only way to prove it will be by trial and error. So, in case this plan doesn’t work, we designed another killing mechanism.

The Kamikaze System

This alternative mechanism consists of the production of a huge amount of lysozyme by the killing cell. This lysozyme in such high concentrations will be able to attack the cell wall of our killer strain of E. coli from the inside out, passing through the inner cell membrane. In consequence, the killer strain will burst, releasing lisozyme into the medium and killing nearby contaminants. That’s why we called it the Kamikaze System. The idea to use this system came from the observations with some E. coli strains used in our lab for heterologous expression. These strains have a basal expression of phage T4 lysozyme, and even in low concentrations minor stresses are able to kill the cells by lysis. For this construct we will use biobricks already in the registry, by fusing a T7 promoter (BBa I7469) designed by the Cambridge 2007 team, to the T4-Endolysin (BBa K112806) designed by the UC Berkeley 2008 team (Fig. 3), but never characterized. Therefore, by characterizing this endolysin we will be helping another iGEM team