Difference between revisions of "Team:SIAT-SCIE/Project Description"

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    <p style="text-align: center;margin-top: 80px"><img src="https://static.igem.org/mediawiki/2018/9/9b/T--SIAT-SCIE--SIAT_Description.png" width="1200px" height="600px"></p>
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<h2>Brief Intro</h2>
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        <h1>Outer Membrane vesicles</h1>
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<p style="font-family:'Avenir';font-size:23px;"> Outer membrane vesicles (OMVs) are ubiquitously produced in the world of bacteria yet have been grievously overlooked in the past; budding out as spherical containers of 20 to 500 nm in diameter from the bacterial membrane, they are potentially capable of transporting a wide array of biomolecules that awaits the academia to divulge. <a href="#r1">[1]</a> As potent transporters, OMVs play an integral role in various biological phenomena, ranging from stress regulation to microbial interactions. <a href="#r2">[2]</a>  
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        <p style="font-size: 20px">Outer membrane vesicles (OMVs) are vesicles produced and used as common vehicles in the world of gram-negative-bacteria. Despite their ubiquity, they have been grievously overlooked in the past; budding out as spherical containers of 20 to 500 nm in diameter from the bacterial membrane, they are potentially capable of transporting a wide array of biomolecules that awaits the academia to divulge. As potent transporters, OMVs play an integral role in various biological phenomena, ranging from stress regulations to microbial interactions (1).
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        <p style="font-size: 20px"> Seeing that the unique properties of OMVs may revolutionise traditional delivery system, our team aims to devise a technique that incorporates the wonders of OMVs and the very frontier technology in genetic engineering — Cas9 proteins — to form a OMV-CRISPR-Cas9 system, which is capable of delivering Cas-9 proteins to target the bacterial genome of bacteria inside mammals’ bodies.
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        <p style="font-size: 20px"> As natural kins to bacterial cell membranes, OMVs can be degraded easily while preserving the shape and bioactivity of sensitive Cas9 proteins within, as well as single guide RNAs (sg-RNAs). We expect this technique would open up new possibilities of in vitro genetic engineering, thus providing substantial aid in curing and preventing illnesses such as inflammatory bowel diseases by removing virulence genes from malignant bacteria with this technique.
 
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<p style="font-family:'Avenir';font-size:23px;">Seeing that the unique properties of OMVs may revolutionize traditional delivery system, our team aims to apply the wonders of OMVs to the very frontiers of genetic engineering research - the CRISPR-Cas9 system. As natural kins to cell membranes, they can be degraded easily while preserving the shape and bioactivity of sensitive Cas9 proteins within, as well as single guide RNA (sg-RNA). We expect this technique would open up new possibilities of in vitro genetic engineering, and be of substantial aid in curing and preventing illnesses such as inflammatory bowel diseases by removing virulence genes from malignant bacteria.</p>
 
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        <h1>Fusobacterium nucleatum
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        <p style="font-size: 20px">Our project aims to test the efficiency of our system by applying it to knock out fadA gene of Fusobacterium nucleatum, a strain of bacteria that reside in a human alimentary canal.
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        <p style="font-size: 20px">F. nucleatum is a gram-negative bacterium prevalently found in mammal oral cavity and is frequently associated with oral inflammation diseases and various cancers (2).  Its virulence stems from its invasion of the human epithelial cells, which induces oncogenic gene expression. The main genetic culprit of its pathogenicity is its fadA gene, which is an adhesion virulence factor that ensures the binding of F.nucleatum to the host epithelial cell, thereby enabling the bacterium to invade the host and causing inflammation and cancer.
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<h2> The Specifics </h2>
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      <p style="font-size: 20px">F. nucleatum’s fadA gene is a congenial candidate for our project since, being gram-negative, F.nucleatum possesses abundant OMVs that are central to our designed system. Through knocking out the fadA gene, we are enabled to observe the efficacy of our system’s application in a realistic setting.
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<p style="font-family:'Avenir';font-size:23px;">As a preliminary step, we will perform transformation of plasmids containing genes of Cas9 and sg-RNA on pathogens, whose efficacy of gene editing may serve as a baseline for further experiments involving OMV transport of the Cas9 protein and sg-RNA.
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<p style="font-family:'Avenir';font-size:23px;">In hopes of maximizing the yield of OMVs, insertion of hypervesiculation genes into the bacteria will be performed. <a href="#r3">[3]</a> A SpyCatcher/SpyTag system will also be constructed with regards to Cas9, which may efficiently direct the protein into the OMV. <a href="#r4">[4]</a>
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<p style="font-family:'Avenir';font-size:23px;">To quantify the expression of Cas9 within the bacteria into which the OMVs have fused, a fluorophore may be attached to the protein for fluorescence analysis.
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<p style="font-family:'Avenir';font-size:23px;">Lastly to confirm the efficacy of our OMV-CRISPR-Cas9 system, the expression of virulence gene in pathogen, which should be mostly removed, may be quantified as well.
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<h2 style="text-align:center"> References </h2>
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<li id='r1'> Liu, Y., Alexeeva, S., Defourny, K. A., Smid, E. J. & Abee, T. (2018). Tiny but mighty: bacterial membrane vesicles in food biotechnological applications. Current Opinion in Biotechnology, 49, 179-184
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<li id='r2'> Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews. Microbiology, 13(10), 605–619.
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<li id='r3'> Valderrama, J. D. & Gutierrez F. R.S. (2018). Lipo Nanocarriers for Drug Targeting. 199-229.
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<li id='r4'>Zakeri, B., Fierer, J. O., Celik, E., Chittock, E. C., Schwarz- Linek, U., Moy, V. T. & Howarth, M. (2012) Peptide Tag Forming a Rapid Covalent Bond to a Protein, Through Engineering a Bacterial Adhesion. Proc. Natl. Acad. Sci. U. S. A. 109, e690−e697. </li>
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Revision as of 16:42, 16 October 2018

Outer Membrane vesicles

Outer membrane vesicles (OMVs) are vesicles produced and used as common vehicles in the world of gram-negative-bacteria. Despite their ubiquity, they have been grievously overlooked in the past; budding out as spherical containers of 20 to 500 nm in diameter from the bacterial membrane, they are potentially capable of transporting a wide array of biomolecules that awaits the academia to divulge. As potent transporters, OMVs play an integral role in various biological phenomena, ranging from stress regulations to microbial interactions (1).

Seeing that the unique properties of OMVs may revolutionise traditional delivery system, our team aims to devise a technique that incorporates the wonders of OMVs and the very frontier technology in genetic engineering — Cas9 proteins — to form a OMV-CRISPR-Cas9 system, which is capable of delivering Cas-9 proteins to target the bacterial genome of bacteria inside mammals’ bodies.

As natural kins to bacterial cell membranes, OMVs can be degraded easily while preserving the shape and bioactivity of sensitive Cas9 proteins within, as well as single guide RNAs (sg-RNAs). We expect this technique would open up new possibilities of in vitro genetic engineering, thus providing substantial aid in curing and preventing illnesses such as inflammatory bowel diseases by removing virulence genes from malignant bacteria with this technique.

Fusobacterium nucleatum

Our project aims to test the efficiency of our system by applying it to knock out fadA gene of Fusobacterium nucleatum, a strain of bacteria that reside in a human alimentary canal.

F. nucleatum is a gram-negative bacterium prevalently found in mammal oral cavity and is frequently associated with oral inflammation diseases and various cancers (2). Its virulence stems from its invasion of the human epithelial cells, which induces oncogenic gene expression. The main genetic culprit of its pathogenicity is its fadA gene, which is an adhesion virulence factor that ensures the binding of F.nucleatum to the host epithelial cell, thereby enabling the bacterium to invade the host and causing inflammation and cancer.

F. nucleatum’s fadA gene is a congenial candidate for our project since, being gram-negative, F.nucleatum possesses abundant OMVs that are central to our designed system. Through knocking out the fadA gene, we are enabled to observe the efficacy of our system’s application in a realistic setting.