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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         <h1>Outer Membrane vesicles</h1>
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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">In our project, we used E.coli BW25113 strain with OmpA gene knocked out for the reason that the absence of OmpA protein would lead to hyper-vesiculation, meaning increased production of outer membrane vesicles (OMVs).</p>
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            <p style="font-size: 20px">Inside the periplasm there is a thin, rigid peptidoglycan(PG) layer attached to both outer membrane and cytoplasmic membrane by membrane anchored proteins.[Fig. 1](1) Deletion or truncation of OmpA, an abundant protein linking the outer membrane and peptidoglycan layer, will therefore result in increased vesiculation in E. coli, Salmonella, and Vibrio cholerae (2) as the Figure 1 lack of OmpA destabilises the periplasm’s structure.This will result in greater likelihood that our Cas9 protein will be encapsulated into OMVs.
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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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        <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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      <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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         <h1>Safety
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         <h1 style="font-size:30px">Importing Proteins into Periplasm Through TAT Pathway
 
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         <p style="font-size: 20px">Out of safety concerns, instead of using the pathogenic Fusobacterium nucleatum, we transform a section of FadA’s coding sequence — with sgRNA’s binding site — into E.coli to test our system’s efficiency.
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         <p style="font-size: 20px">To allow proteins to be encapsulated in OMVs, they need to be imported into bacteria’s periplasm first. Compared to other common transporting pathways that transport proteins before folding (e.g. Sec and SRP-dependent pathways), twin arginine transport (TAT) pathway is an available option for transportation of fully folded proteins(3). This is crucial since some proteins, including GFP, cannot fold properly when transported into periplasm(3).  
 
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        <p style="font-size: 20px">Thus, they have to complete their folding in cytoplasm before they can be transported to periplasm. Consequently, TAT pathway is the only plausible option[Fig. 2]. Regarding the Cas9 proteins, since there has been no literature discussing the possibility of their possible
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            Figure 2 of folding in periplasm, we chose TAT pathway for Cas9 proteins as well out of prudence.
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            <summary>Testing TorA Signal Peptide Efficiency</summary>
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            <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;">TorA is one of the signal peptides that trigger TAT pathway. The addition of TorA to the recombinant proteins can enable the transportation of protein to periplasm.</p>
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              <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;">In order to test TorA’s working efficiency, we first fused it with GFPmut3 for more convenient reading. Then we transformed plasmids with TorA-GFP into E.coli.  After cultivating the transformed E.coli overnight, osmotic shock assay would be carried out to separate cytoplasm and periplasm, thereby enabling a direct reading of the latter as high degree of fluorescent light in the periplasm indicates the robustness and efficiency of TorA.
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                <h3>Four sets of samples are needed:
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                <li>WT with GFP
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                <li>ΔOmpA with GFP
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                </li>
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                <li>WT with TorA-GFP
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                </li>
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                <li>ΔOmpA with TorA-GFP
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Revision as of 05:55, 17 October 2018

Chassis

In our project, we used E.coli BW25113 strain with OmpA gene knocked out for the reason that the absence of OmpA protein would lead to hyper-vesiculation, meaning increased production of outer membrane vesicles (OMVs).

Inside the periplasm there is a thin, rigid peptidoglycan(PG) layer attached to both outer membrane and cytoplasmic membrane by membrane anchored proteins.[Fig. 1](1) Deletion or truncation of OmpA, an abundant protein linking the outer membrane and peptidoglycan layer, will therefore result in increased vesiculation in E. coli, Salmonella, and Vibrio cholerae (2) as the Figure 1 lack of OmpA destabilises the periplasm’s structure.This will result in greater likelihood that our Cas9 protein will be encapsulated into OMVs.

Importing Proteins into Periplasm Through TAT Pathway

To allow proteins to be encapsulated in OMVs, they need to be imported into bacteria’s periplasm first. Compared to other common transporting pathways that transport proteins before folding (e.g. Sec and SRP-dependent pathways), twin arginine transport (TAT) pathway is an available option for transportation of fully folded proteins(3). This is crucial since some proteins, including GFP, cannot fold properly when transported into periplasm(3).

Thus, they have to complete their folding in cytoplasm before they can be transported to periplasm. Consequently, TAT pathway is the only plausible option[Fig. 2]. Regarding the Cas9 proteins, since there has been no literature discussing the possibility of their possible Figure 2 of folding in periplasm, we chose TAT pathway for Cas9 proteins as well out of prudence.

Testing TorA Signal Peptide Efficiency

TorA is one of the signal peptides that trigger TAT pathway. The addition of TorA to the recombinant proteins can enable the transportation of protein to periplasm.

In order to test TorA’s working efficiency, we first fused it with GFPmut3 for more convenient reading. Then we transformed plasmids with TorA-GFP into E.coli. After cultivating the transformed E.coli overnight, osmotic shock assay would be carried out to separate cytoplasm and periplasm, thereby enabling a direct reading of the latter as high degree of fluorescent light in the periplasm indicates the robustness and efficiency of TorA.

Four sets of samples are needed:

  • WT with GFP
  • ΔOmpA with GFP
  • WT with TorA-GFP
  • ΔOmpA with TorA-GFP