Team:Auckland New Zealand/Project

From 2014.igem.org

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<td > <h3> Project Overview </h3></td>
<td > <h3> Project Overview </h3></td>
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<p>Numerous studies have  suggested probiotic skin flora utilize various mechanisms to inhibit pathogenic infection, such as competition to binding sites, nutrients, production of inhibitory bacteriocins and organic acids that kill or inhibit growth of  the pathogens. We aim to engineer a bacterial strain which, when applied to a wound site, will prevent the infection of the wound, such as by ''Staphylococcus aureus'', a common skin pathogen.
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<p>Numerous studies have  suggested probiotic skin flora utilize various mechanisms to inhibit pathogenic infection, such as competition to binding sites, nutrients, production of inhibitory bacteriocins and organic acids that kill or inhibit growth of  the pathogens. We aim to engineer a bacterial strain which, when applied to a wound site, will prevent the infection of the wound, such as by Staphylococcus aureus, a common skin pathogen.
To achieve this the engineered bacterial strain will incorporate the following components:
To achieve this the engineered bacterial strain will incorporate the following components:
</p>
</p>
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<td > <h3> Project Details </h3></td>
<td > <h3> Project Details </h3></td>
<p>
<p>
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We will focus on the Biological Protection aspect of Bac-Barrier in the 2014 iGEM project. Instead of the usual methods of devising mechanisms to secrete antimicrobial substances to kill of incoming pathogens, we have chosen to approach this in a rather unorthodox method - We will devise mechanisms which prevent the pathogenic bacteria reaching the wound, instead of killing them outright. The reasoning for this is that within a species of pathogenic bacteria, there will be natural variation in the susceptibility to antimicrobial substances. The usage of antibiotics will facilitate the selection of resistant strains, rendering the long term usage ineffective.
+
We will focus on the Biological Protection aspect of Bac-Barrier in the 2014 iGEM project. Instead of the usual methods of devising mechanisms to secrete antimicrobial substances to kill of incoming pathogens, we have chosen to approach this in a rather unorthodox method - We will devise mechanisms which prevent the pathogenic bacteria reaching the wound, instead of killing them outright. The reasoning for this is that within a species of pathogenic bacteria, there will be natural variation in the susceptibility to antimicrobial substances. The usage of antibiotics will facilitate the selection of resistant strains, rendering the long term usage of the antibiotic ineffective.
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In contrast, if the pathogens are unable to access the wound site, they would not be able to cause infection, whether they are present or not.
 +
</p>
 +
<p>
 +
We aim to use attachment factors to make the engineered bacteria occupy potential binding sites of pathogens, and to bind to each other, creating a literal microbial barrier.
 +
</p>
 +
<td > <h3> Materials and Methods </h3></td>
 +
<p>
 +
We have decided to use the Fibronectin Binding Protein A (FbpA) to facilitate adherence to the wound site and Human Epithelial Cadherin (E-Cadherin) to facilitate binding to each other. These proteins will have to be expressed on the outer membrane of the bacterial cells to have a function. To this end, we are using an existing Biobrick for surface expression (Part:BBa_K811005): the truncated ice nucleation protein (INP) with multiple cloning sites. We eventually aim to test the ability of the engineered bacteria to prevent S. aureus infection by exposing human epithelial cell lines colonized by Bac-Barrier to ''S. aureus'' and recording the extent of S. aureus colonization compared to human epithelial cells which were not colonized by Bac-Barrier.
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</p>
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<td > <h3> The Experiments </h3></td>
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<p>
 +
Due to unforeseen circumstances, the gene for FbpA and E-Cadherin is still in synthesis. As such, we were not able to submit the Biobrick parts on time, but we still aim to submit them to the registry when they are completed.
 +
</p>
 +
<p>
 +
For the gene to be expressed, a promoter and a ribosome binding site is needed, as well as the outer surface expression platform. The promoter used is Part:BBa_J23100, the RBS is Part:BBa_B0034 and the expression platform used is Part:BBa_K811005. Part:BBa_J23100 and Part:BBa_B0034 was in the distribution kit and E. coli was transformed with the relevant plasmids. Part:BBa_K811005 was received as an agar stab from iGEM HQ and was streaked onto a Lysogeny Broth (LB) plate containing chloramphenicol.
 +
</p>
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<td > <h3> Results </h3></td>
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<p>
 +
We will likely not be able to upload the results because of the wiki freeze on 17/10/2014, but will present our case at the Jamboree is possible.
 +
</p>
 +
<td > <h3> Data analysis </h3></td>
 +
We will likely not be able to upload the data analysis because of the wiki freeze on 17/10/2014, but will present our case at the Jamboree is possible.
 +
<p>
 +
</p>
 +
<td > <h3> Conclusions </h3></td>
 +
<p>
 +
We will likely not be able to upload the data analysis because of the wiki freeze on 17/10/2014, but will present our case at the Jamboree is possible. However, we expect that colonization by Bac-Barrier will result in a decrease in subsequent S. aureus infection.
</p>
</p>
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<ol>
 
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<li>'''Project Overview'''</li>
 
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<li>Project Details</li>
 
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<li>Materials and Methods</li>
 
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<li>The Experiments</li>
 
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<li>Results</li>
 
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<li>Data analysis</li>
 
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<li>Conclusions</li>
 
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</ol>
 
<h3>References </h3>
<h3>References </h3>
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iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you. </p>  
iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you. </p>  
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<p> You can use these subtopics to further explain your project</p>
 
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Revision as of 23:46, 13 October 2014



Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions

Bac-Barrier (Name subject to change)

Overview

With the increase of antibiotic resistance in pathogenic microorganisms, the management of cutaneous injuries with traditional methods (such as bandaging and antibiotic application) has become less effective. This project will introduce the concept of selective colonization of the wound site by an engineered microorganism to prevent subsequent infection by pathogens.


Project Overview

Numerous studies have suggested probiotic skin flora utilize various mechanisms to inhibit pathogenic infection, such as competition to binding sites, nutrients, production of inhibitory bacteriocins and organic acids that kill or inhibit growth of the pathogens. We aim to engineer a bacterial strain which, when applied to a wound site, will prevent the infection of the wound, such as by Staphylococcus aureus, a common skin pathogen. To achieve this the engineered bacterial strain will incorporate the following components:

(1) Regulatory control - It must have mechanisms which will restrict the growth and survival of the engineered microbe to designated sites and avoid unwanted spread. This is achieved by a suicide switch which is regulated by the presence/absence of signal substances.

(2)Biological Protection - It will have mechanisms which will inhibit the growth of other exogenous microorganisms. This will be achieved by blocking the binding sites on the wound as well as spatial exclusion of the exogenous bacteria to the wound site.

(3) Mechanical Protection - It will incorporate mechanisms which will protect the wound from further physical damage as well as from other factors such as dessication. Biofilm formation is being investigated as to whether it will suit this purpose.

(4)It will contain mechanisms which assists in wound healing. Such mechanisms could be factors which induce keratinocyte proliferation or factors which reduces/stop bleeding.

Project Details

We will focus on the Biological Protection aspect of Bac-Barrier in the 2014 iGEM project. Instead of the usual methods of devising mechanisms to secrete antimicrobial substances to kill of incoming pathogens, we have chosen to approach this in a rather unorthodox method - We will devise mechanisms which prevent the pathogenic bacteria reaching the wound, instead of killing them outright. The reasoning for this is that within a species of pathogenic bacteria, there will be natural variation in the susceptibility to antimicrobial substances. The usage of antibiotics will facilitate the selection of resistant strains, rendering the long term usage of the antibiotic ineffective. In contrast, if the pathogens are unable to access the wound site, they would not be able to cause infection, whether they are present or not.

We aim to use attachment factors to make the engineered bacteria occupy potential binding sites of pathogens, and to bind to each other, creating a literal microbial barrier.

Materials and Methods

We have decided to use the Fibronectin Binding Protein A (FbpA) to facilitate adherence to the wound site and Human Epithelial Cadherin (E-Cadherin) to facilitate binding to each other. These proteins will have to be expressed on the outer membrane of the bacterial cells to have a function. To this end, we are using an existing Biobrick for surface expression (Part:BBa_K811005): the truncated ice nucleation protein (INP) with multiple cloning sites. We eventually aim to test the ability of the engineered bacteria to prevent S. aureus infection by exposing human epithelial cell lines colonized by Bac-Barrier to ''S. aureus'' and recording the extent of S. aureus colonization compared to human epithelial cells which were not colonized by Bac-Barrier.

The Experiments

Due to unforeseen circumstances, the gene for FbpA and E-Cadherin is still in synthesis. As such, we were not able to submit the Biobrick parts on time, but we still aim to submit them to the registry when they are completed.

For the gene to be expressed, a promoter and a ribosome binding site is needed, as well as the outer surface expression platform. The promoter used is Part:BBa_J23100, the RBS is Part:BBa_B0034 and the expression platform used is Part:BBa_K811005. Part:BBa_J23100 and Part:BBa_B0034 was in the distribution kit and E. coli was transformed with the relevant plasmids. Part:BBa_K811005 was received as an agar stab from iGEM HQ and was streaked onto a Lysogeny Broth (LB) plate containing chloramphenicol.

Results

We will likely not be able to upload the results because of the wiki freeze on 17/10/2014, but will present our case at the Jamboree is possible.

Data analysis

We will likely not be able to upload the data analysis because of the wiki freeze on 17/10/2014, but will present our case at the Jamboree is possible.

Conclusions

We will likely not be able to upload the data analysis because of the wiki freeze on 17/10/2014, but will present our case at the Jamboree is possible. However, we expect that colonization by Bac-Barrier will result in a decrease in subsequent S. aureus infection.

References

iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you.

It's important for teams to describe all the creativity that goes into an iGEM project, along with all the great ideas your team will come up with over the course of your work.

It's also important to clearly describe your achievements so that judges will know what you tried to do and where you succeeded. Please write your project page such that what you achieved is easy to distinguish from what you attempted.