Team:UNICAMP-Brazil/Coliguard/Killing

From 2009.igem.org

(Difference between revisions)
Line 1: Line 1:
{{:Team:UNICAMP-Brazil/inc_topo}}
{{:Team:UNICAMP-Brazil/inc_topo}}
-
==The Coliguard - Killing==
+
=The Coliguard - Killing=
-
'''Introduction'''
+
==Introduction==
-
+
-
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.
+
After the detection of a contaminant by our Coli Guard 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 Lactobacilli, 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, Lactobacilli.
 +
 +
The main difference is that E. coli is a Gram Negative bacteria and Lactobacilli is a Gram positive. That means E. coli have two cell membranes around  a thin cell wall witch 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 Lactobacilli 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 (Binet et. Al, 1997).
 +
 
 +
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 (Holland et. Al, 1990, Gentschev et. Al 1996)
 +
 
 +
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.
 +
 
 +
[[Image:Imagem11.jpg|center|500px]]
 +
 
 +
 
 +
We will fuse to this biobrick another one with the lambda phage’s lysozyme without the stop codon, Picture2. We hope this lysozyme with the signaling peptide will be secreted and outside the cell 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 another killing mechanism.
 +
 
 +
==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 (Young 1992)
 +
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 a 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 other wise both lethal and antidote gene will be transferred to the contaminant and the all 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 (James et al. 1996, Braun et al. 2004)
 +
 
 +
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 (Kleanthous et al. 2002)
 +
 
 +
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.
 +
 
 +
==References==
 +
Binet, R.,  Letoffe, S., Ghigo, J.M., Delepelaire, P.,  Wandersman, C. Gene,  1997, 192, 7–11.
 +
 
 +
Braun, V., Pilsl, H. and Gross, P. Arch. Microbiol. 2004. 161, 199–206.
 +
 
 +
Gentschev I., Mollenkopf H., Sokolovic Z., Hess J., Kaufmann S.H.E., Goebel W. Gene. 1996, 179,133–140.
 +
 
 +
Holland, I.B., Blight, M.A. and Kenny, B. J. Bioenerg. Biomembr. 1990. 22,  473 491.
 +
 
 +
James, R., Kleanthous, C. and Moore, G.R.,  Microbiology.1996. 142, 1569–1580.
 +
 
 +
Kleanthous, C., Hemmings, A.M., Moore, G.R. and James, R. Mol. Microbiol.. 2002. 28, 227–233.
 +
 
 +
Young R. Microbiol Rev. 1992,  56. 430–481.
-
'''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
 
{{:Team:UNICAMP-Brazil/inc_rodape}}
{{:Team:UNICAMP-Brazil/inc_rodape}}

Revision as of 20:57, 21 October 2009

Topo l2.gif topo_r_igem.gif
topo_r_b.gif

Contents

The Coliguard - Killing

Introduction

After the detection of a contaminant by our Coli Guard 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 Lactobacilli, 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, Lactobacilli.

The main difference is that E. coli is a Gram Negative bacteria and Lactobacilli is a Gram positive. That means E. coli have two cell membranes around a thin cell wall witch 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 Lactobacilli 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 (Binet et. Al, 1997).

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 (Holland et. Al, 1990, Gentschev et. Al 1996)

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.

Imagem11.jpg


We will fuse to this biobrick another one with the lambda phage’s lysozyme without the stop codon, Picture2. We hope this lysozyme with the signaling peptide will be secreted and outside the cell 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 another killing mechanism.

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 (Young 1992) 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 a 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 other wise both lethal and antidote gene will be transferred to the contaminant and the all 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 (James et al. 1996, Braun et al. 2004)

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 (Kleanthous et al. 2002)

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.

References

Binet, R., Letoffe, S., Ghigo, J.M., Delepelaire, P., Wandersman, C. Gene, 1997, 192, 7–11.

Braun, V., Pilsl, H. and Gross, P. Arch. Microbiol. 2004. 161, 199–206.

Gentschev I., Mollenkopf H., Sokolovic Z., Hess J., Kaufmann S.H.E., Goebel W. Gene. 1996, 179,133–140.

Holland, I.B., Blight, M.A. and Kenny, B. J. Bioenerg. Biomembr. 1990. 22, 473 491.

James, R., Kleanthous, C. and Moore, G.R., Microbiology.1996. 142, 1569–1580.

Kleanthous, C., Hemmings, A.M., Moore, G.R. and James, R. Mol. Microbiol.. 2002. 28, 227–233.

Young R. Microbiol Rev. 1992, 56. 430–481.