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| − | <p style="text-align: center;margin-top: 80px"><img src="https://static.igem.org/mediawiki/2018/0/09/T--SIAT-SCIE--SIAT_Design.png" width="1200px" height="600px"></p> | + | <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> |
| | <div class="chassis"> | | <div class="chassis"> |
| − | <h1>Chassis</h1> | + | <h1>Outer Membrane vesicles</h1> |
| − | <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> | + | <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).</p> |
| | | | |
| − | <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. | + | |
| | + | <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/3/36/T--SIAT-SCIE--SIAT_OMV%281%29.png" width="800px" height="400px"></p> |
| | + | <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. |
| | </p> | | </p> |
| − | <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/9/95/T--SIAT-SCIE--Chassis_Figure1.png" width="600px" height="600px"></p> | + | <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.</p> |
| | + | |
| | + | <h1>Fusobacterium nucleatum</h1> |
| | + | <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 human alimentary canal. </p> |
| | + | <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 insures the binding of F.nucleatum to the host epithelial cell, thereby enabling the bacterium to invading the host and causing inflammation and cancer. </p> |
| | + | <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 realistic setting.</p> |
| | + | <p style="font-size: 20px"></p> |
| | </div> | | </div> |
| − | <div class="chassis">
| + | |
| − | <h1 style="font-size:30px">Importing Proteins into Periplasm Through TAT Pathway
| + | |
| − | </h1>
| + | |
| − | <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).
| + | |
| − | </p>
| + | |
| − | <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
| + | |
| − | Figure 2 of folding in periplasm, we chose TAT pathway for Cas9 proteins as well out of prudence.
| + | |
| − | </p>
| + | |
| − | <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/d/d3/T--SIAT-SCIE--Chassis_Figure2.png" width="500px" height="600px"></p>
| + | |
| − | </div>
| + | |
| − | <section style="margin-top:-130px">
| + | |
| − | | + | |
| | <details> | | <details> |
| − | <summary>Testing TorA Signal Peptide Efficiency</summary> | + | <summary>Mechanism of FadA protein</summary> |
| − | <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;margin:40px 200px 0px 200px">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> | + | <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;margin:40px 200px 0px 200px">The FadA protein is activated when its two forms combine and become internalized. The first form is a pre-FadA that is anchored in the cell membrane, whereas the second form is the mature FadA (mFadA) that is secreted out of F. nucleatum. When the two forms combine to form a complex, the protein is capable to help F. nucleatum bind to the host epithelial cell, thus allowing F. nucleatum embark on invading the host cells.</p> |
| − | <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;margin:0px 200px 0px 200px">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. | + | <h3 style="margin: 20px 200px 30px 200px;font-size:30px">Overview (Fig. 1)</h3> |
| | + | <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;margin:0px 200px 0px 200px">We will first express Cas9 and sgRNA in E.coli and then transport them to E.coli’s periplasm(Step 1). By then, they may be packaged by OMVs that bud off from E.coli’s outer membrane(Step 2). Those OMVs will be collected and mixed with our target bacteria, thereby allowing them to fuse with bacteria again, to release the Cas9 proteins and sgRNA(Step 3), and cleave the target gene (Step 4 & 5). Afterwards, we will test whether the target gene is cleaved by Cas9. |
| | </p> | | </p> |
| − | <div class="chassis" style="padding-left: 200px">
| + | <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/3/30/T--SIAT-SCIE--SIAT_Description_Figure1.png" height="600px" width="900px"></p> |
| − | <h3>Four sets of samples are needed:
| + | |
| − | </h3>
| + | |
| − | <ul>
| + | |
| − | <li>WT with GFP
| + | |
| − | </li>
| + | |
| − | <li>ΔOmpA with GFP
| + | |
| − | </li>
| + | |
| − | <li>WT with TorA-GFP
| + | |
| − | </li>
| + | |
| − | <li>ΔOmpA with TorA-GFP
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;margin:0px 200px 0px 200px;line-height: 25px">We used plate reader to measure the fluorescence value of both periplasmic and cytoplasmic fractions and calculate their fluorescent ratio (periplasm : cytoplasm) in order to take background noises into account. We expect the ratio to be approximately sample 3＞sample 4＞sample 2≈sample1, for the bacteria with TorA-GFP should have a larger amount of GFP in periplasm. Lower fluorescence reading in sample 4 than sample 3 indicates higher yield of OMVs in ΔOmpA strain as more GFP are encapsulated by OMVs and leave periplasm.
| + | |
| − | </p>
| + | |
| | </details> | | </details> |
| | </article> | | </article> |
| − | </section>
| + | |
| − | <div class="chassis">
| + | |
| − | <h1>CjCas9</h1>
| + | |
| − | <p style="font-size: 20px;">Since OMVs generally have a limited capacity as they only have a size ranging from 50nm to 200nm, proteins should be as small as possible for them to be encapsulated into OMVs. Recently characterized cjCas9(derived from Campylobacter.jejuni), with a sequence length of 2.95Kbp making it one of the smallest Cas9 orthologues, became our first choice. Moreover, cjCas9 has a high specificity, cleaving only a limited and particular number of sites in human and mouse genome (4), thereby making it a safer candidate for its application in mammals..</p>
| + | |
| − | </div>
| + | |
| − | <section style="margin: 20px;padding-top:0px">
| + | |
| − | <article>
| + | |
| − | <details>
| + | |
| − | <summary>sgRNA</summary>
| + | |
| − | <h2>PAM</h2>
| + | |
| − | <p style="font-size: 20px;line-height: 25px">Due to conflicting reports on the PAM sequence of cjCas9, we adopted the most up-to-date results: 5‘-NNNVRYM-3’ PAM with the preference for T and C at positions 6 and 7 (5). There are many ‘PAM’ sites in FadA. Among them, we chose the one closest to 5’ as the cleaving site, in hope that the introduction of indels caused by cjCas9’s cleavage should disrupt most of FadA’s reading frame, rendering it non-functional.
| + | |
| − | </p>
| + | |
| − | <h2>crRNA</h2>
| + | |
| − | <p style="font-size: 20px;line-height: 25px">The CRISPR guide RNA (crRNA) is a type of RNA that enables CRISPR proteins to locate their targets. For an enhanced efficacy and on-target rate at knocking out fadA by our cjCas9, the design of crRNA is very important. Unlike most of Cas9 orthologues, cjCas9 recognizes the PAM nucleotides on both the target and non-target DNA strands. Our crRNA is designed to recognized the PAM strand.</p>
| + | |
| − | <p style="font-size: 20px;line-height: 25px">We copy the rest of the sgRNA[Fig.3] sequence according to Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.</p>
| + | |
| − | <p style="text-align:center"><img src="https://static.igem.org/mediawiki/2018/9/96/T--SIAT-SCIE--Chassis_Figure3.png" width="600px" height="500px"> </p>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>Testing cjCas9’s cleavage efficiency
| + | |
| − | </summary>
| + | |
| − | <p style="font-size: 20px;margin-top:20px;line-height: 25px">After cjCas9 recognizing the cleavage site on FadA gene, a double-stranded break shall be introduced to the fadA gene, thus triggering DNA repair through nonhomologous end joining(NHEJ)[Fig.4]. NHEJ causes nearly random insertion and deletion mutations (i.e. indels) at the double-stranded break site, and thus leading to gene knockout (e.g., by causing a shift in the target gene’s reading frame or mutating a critical region of the encoded protein) (6).</p>
| + | |
| − | <p style="font-size: 20px;line-height: 25px">To detect FadA’s frameshift, we fused a part of FadA with a mRFP protein using a GILQSTVPRARNP linker (7), creating a FadA-linker-mRFP segment. If cjCas9 successfully cleaved mRFP and resulted in a frameshift, mRFP would cease to functioning, thereby weakening the fluorescence. Subsequently, sequencing was carried out to test if cjCas9 did cause a mutation that does not affect the reading frame.</p>
| + | |
| − | <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/e/ef/T--SIAT-SCIE--Chassis_Figure4.png" width="700px" height = "450px"></p>
| + | |
| − | </details>
| + | |
| − | <details>
| + | |
| − | <summary>Biobrick</summary>
| + | |
| − | <p style="line-height: 30px;font-size: 25px;padding-top:20px;margin-top: 30px;line-height: 30px"><span class="BoldLarge">CjCas9</span>[Fig. 5] is under the control of ptet-tetR promoter. The backbone consists of a pSC101 origin (yellow) and a Chloramphenicol resistant gene (light green).</p>
| + | |
| − | <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/5/5d/T--SIAT-SCIE--Chassis_Figure5.png" height="750px" width="750px"></p>
| + | |
| − | <p style="line-height: 30px;font-size: 25px;padding-top:20px;margin-top: 30px;line-height: 30px"><span class="BoldLarge">sgRNA</span>[Fig. 6] is under the control of J23100 promoter. It is inserted in pSB1A3 vector.</p>
| + | |
| − | <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/2/21/T--SIAT-SCIE--Chassis_Figure6.png" height="250px" width="750px"></p>
| + | |
| − | </details>
| + | |
| − | </article> | + | |
| − | </section>
| + | |
| − |
| + | |
| | </body> | | </body> |
| | </html> | | </html> |
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 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 insures the binding of F.nucleatum to the host epithelial cell, thereby enabling the bacterium to invading 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 realistic setting.
