Team:UNICAMP-Brazil/Coliguard/Killing

=The Coliguard - Killing=

Introduction
After the detection of a contaminant by our Coliguard System, part of the labor population must become the killer population, which has as the most remarkable feature the ability to kill this contaminant.

As our project is focused to solve the problem in the production of ethanol, we decided to develop our killing mechanism to be able to destroy the most important bacterial contaminant of this process, the Lactobacillus group, a group of Gram positive bacteria.

But how can a bacteria kill another bacteria without killing itself? That was our first question to answer in the development of this mechanism. To solve this problem we focused in the main biological difference between our guard bacteria, E. coli, and the contaminant, Lactobacillus. The main difference is that E. coli is a Gram Negative bacteria and Lactobacillus is a Gram positive. That means E. coli have two cell membranes around a thin cell wall which remains isolated from the intracellular and extracellular environment. That not occurs with the Gram positive bacteria, because they have a thick cell wall with direct contact with the extracellular environment and surrounds the cell membrane.

With these differences in mind we propose a way to attack the exposed cell wall of Lactobacillus secreting something in the medium capable of doing harm just to this structure and not to E. coli’s outer membrane. We found lysozymes the most able enzyme to do this work, and we chose it as our weapon.

Now that our weapon has been chosen, we face a new problem. How to put it out the E. coli?

The Alpha Hemolysin Secretion System
E. coli, as a Gram negative bacteria, doesn’t have a well developed secretion system to transport proteins to the extracellular medium. The best system we found is the alpha hemolysin secretion system.

The alpha hemolysin secretions system is encoded in a operon containing four genes, Picture1A: hlyD and HlyB constitutes the transporter, HlyA is the hemolysin itself and HlyC codifies to a protein important to make HlyA active (1).

To use the hemolysin system we intend to construct a biobrick with HlyB and HlyD and 252 bp of the carboxy terminal region of HlyA, using primers in Silver Standard, Picture 1B. This 252 bp works as a signaling region enabling the genes fused to it to codify for a peptide capable of being recognized by hlyB and hlyD and be transported outside the cell (2,3).

This is the first biobrick designed to make E. coli secretes a protein using a transport system and can be used to a big range of targets helping to solve the problem of secretion in E. coli.



We will fuse to this biobrick another one with the lambda phage’s lysozyme without the stop codon. We hope this lysozyme with the signaling peptide will be secreted and outside the cell it will be able to kill and destroy all the Lactobacilli and Gram positive contaminants who dare to stay on our way.

This secretion system doesn’t works for all proteins and the only way to know it is by trial and error. So in parallel we created others killing mechanisms.

The Kamikaze System
The other mechanism consist in the production of a huge amount of lysozyme by the killing cell, this lysozyme in high concentrations will be able to attack the cell wall of E. coli passing trough the inner cell membrane. This will destroy the E. coli releasing lisozyme in the medium, that’s why we called it The Kamikaze System (4). The idea to use this system came from the observations with some E. coli strains used in our lab for heterologous expression, this strains have a basal expression of phage T4 lysozyme and even in low concentrations with minor stress the cell lysis.

To test this device we will use only biobricks already made, we will fuse a T7 promoter, BBa I7469, designed by the Cambridge 2007 team, to the T4-Endolysin, BBa K112806, designed by the UC Berkeley 2008 team.



The Colicin System
To improve our killing mechanism, giving to it more precision, we developed a third killing mechanism. The two killing mechanism cited before can be very effective and act to contaminants situated far away from the killing, but these system create a problem: The dilution of the lysozymes in space and consequent reducing of the killing effectiveness.

To solve this problem we focused on the closest relation two different bacteria can have, the conjugation. The conjugation is already being used in our project to detect the contaminants, but we can improve the applicability of it making the conjugation not only to detect the contaminants but also to kill them.

If we insert in the F plasmid a gene that codifies to a lethal protein, after the transfer of DNA the contaminant will produces this protein and kill itself.

Our Killer bacteria must have an antidote to the lethal protein to avoid its suicide and the antidote gene must be in the chromosomal DNA while the lethal gene must be in the F plasmid, otherwise both lethal and antidote gene will be transferred to the contaminant and the entire system will be useless.

As the lethal gene we chose CeaB, which codifies to the colicin E2 that acts as a endonuclease and the antidote we chose CeiB which codifies to a immunity protein against CeaB (5, 6)

This both genes are found in wild populations of E. coli, when a cell harbouring a plasmid with colicin operon dies it releases colicin E2 which is transported inside others neighbors cells. If the cell that receives the colicin has the CeiB immunity protein, no problem, but if it don’t have, the colicin E2 will degrade the cell’s DNA and kill it (7).

In our system, only the contaminant which receive CeaB will transcript and translate this gene leading to its death by the destruction of its own DNA. This system is less metabolic expensive than the Alpha Hemolysin Secreation System and the Kamikaze Sytem, the target will have to afford with the costs of his own killing system.