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From 2014.igem.org

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Bac-Barrier

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 hope to submit them to the registry when they are completed, if the circumstances allow.

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 are unable to upload the results by the date of the wiki freeze on 17/10/2014.

Data analysis

We are unable to upload the results by the date of the wiki freeze on 17/10/2014.

Conclusions

We are unable to upload the results by the date of the wiki freeze on 17/10/2014. However, we expect that colonization by Bac-Barrier will result in a decrease in subsequent S. aureus infection.

References

Initial inspiration for the project: Sikorska H, Smoragiewicz W. (2013). Role of probiotics in the prevention and treatment of meticillin-resistant Staphylococcus aureus infections. Int J Antimicrob Agents. 42:475-481. doi: 10.1016/j.ijantimicag.2013.08.003.

Study that encapsulates the essence of what we are trying to achieve: Prince T, McBain AJ, O'Neill CA.(2012). Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion. Appl Environ Microbiol. 78:5119-5126. doi: 10.1128/AEM.00595-12.