Difference between revisions of "Team:Hawaii/Description"

 
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        <h1>Description</h1>
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    <h1>TO THE CENTROMERE</h1>
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<h2> What Are VLPs?</h2>
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    <h2>with retrotransposon VLPs</h2>
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        <p>VLPs are non-infectious virus-like particles that resemble the shell or capsid of a virus, but do not contain any viral genetic material. VLPs are a hot topic in research because of their many applications. VLPs also provide scientists with a safe way to research important but dangerous viruses. Our team is working with a VLP made from Gag polyproteins. The Gag polyprotein we are working with codes for a capsid, nucleocapsid, protease, reverse transcriptase, and integrase. All these pieces allow us to create VLPs.</p>
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    <p>Centromere retrotransposon (CR) elements offer a natural transportation system to carry genes to the centromere. These elements
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<br>
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      utilize virus-like particles to encapsulate their genome for reverse transcription, then reintegration into the centromere.
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<h2>Retrotransposons!</h2>
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      Centromeres as a transgene target allows for the accumulation of traits due to lack of recombinatorial events. We explored
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<p>Our VLP is derived from a centromeric retrotransposon found in Zea mays. Retrotransposons are segments of RNA that are known for their ability to “jump” to new places in an organism’s genome. Our centromeric retrotransposon is part of the Ty3-Gypsy superfamily. Our team has decided to work with centromeric retrotransposons for their unique ability to target and insert DNA in and near centromeres.</p>
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      the virus-like particle vehicle in this system and measured the stability of the structure for packaging molecular cargo.</p>
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<br>
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    <a href="#abstract">See our abstract.</a>
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<h2>Our Project</h2>
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  </div>
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<p>Our aim is to create a standardized VLP that would act as the perfect “box” to package genetic material or even small proteins. Because this particular Gag polyprotein is not well understood, we are striving to make contributions that will help other teams and researchers understand the nature of this polyprotein and the VLPs they create. </p>
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<p>The first part of our project consists of conducting experiments to determine the conditions in which our protease actively cleaves the Gag polyprotein, and whether our Gag polyprotein self-assembles into VLPs in E. coli. Past studies suggest that Gag polyproteins from the Ty3-Gypsy family self-assemble into VLPs using E. coli as the chassis (1). By using electron microscopy, we can determine whether our VLP self-assembles in E. coli, or if in vitro methods are needed to create VLPs.</p>
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<p>The second aim of our project is to create a system to verify whether VLPs have been formed without complete dependence on electron microscopy. Using a split-protein system with fluorescent proteins or ß-galactosidase would allow us to verify VLP formation. Attaching two halves of GFP to our Gag polyprotein so that these halves only connect when the VLP is fully formed would allow us to qualitatively verify that we have achieved VLP formation, without the use of electron microscopy. This is an important step in creating a VLP that is easy to use.</p>
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<p>Our final challenge is creating a VLP that can efficiently package nucleic acids and proteins. Past studies show it is possible to package proteins within VLPs using a clever RNA aptamer, or a short RNA sequence that binds to the targeted molecule (2). Working to elucidate the RNA sequence our nucleocapsid binds to will allow us to create our own aptamer in which we can then have a standardized way to package targeted molecules.
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</p>
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<p>Our hope for this project is that we create an easy-to-use, standardized VLP that can efficiently package nucleic acids and proteins. A standardized VLP could allow us and future iGEM teams to package a variety of molecules without having to alter the VLP itself.</p>
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<br>
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<h2>Real World Applications</h2>
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<p>Since our VLP is derived from a centromeric retrotransposon, it has the ability to target the centromeres of our organism. Many plant transformation and gene transfer methods have been highly studied and continue to be improved. Gene transfer in many plants at efficient enough rates continues to be a difficult issue to tackle (3). Using our VLP to deliver genetic material to the centromere of the targeted plant could result in high retention of our inserted DNA through many generations, since DNA in and near centromeres are highly conserved in many organisms.</p>
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<br>
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<h2>References</h2>
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<p>1. Larsen LSZ, Kuznetsov Y, McPherson A, Hatfield GW, Sandmeyer S. TY3 GAG3 protein forms ordered particles in Escherichia coli. Virology. 2008;370(2):223-227. doi:10.1016/j.virol.2007.09.017</p>
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<p>2. Fiedler JD, Brown SD, Lau JL, Finn MG. RNA-Directed Packaging of Enzymes within Virus-like Particles **. Angew Chemie Int Ed. 2010;49:9648-9651. doi:10.1002/anie.201005243
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</p>
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<p>3. Potrykus I. Gene Transfer to Plants: Assessment of Published Approaches and Results. Assessment. 1991:205-225. doi:10.1146/annurev.pp.42.060191.001225</p>
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<!--<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
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    <h1>NATURAL VLP FORMATION</h1>
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    <h2>using bioinformatics and a protease assay</h2>
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    <p>To determine the most natural construct for VLP formation, we needed to identify the protease cut site that would allow for mature VLP formation. Initial research on aspartyl proteases allowed us to determine putative protease cleavage sites between hydrophobic amino acid residues.</p>
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    <p>To experimentally determine the protease cleavage sites, we transformed E.coli with a construct containing the gag and protease domains. Following induction, autocatalytic protease activity was observed and the resulting protein bands were sent to the Taplin Mass Spectrometry
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    Facility. Tryptic fragments confirmed our previous putative cut site downstream of the nucleocapsid.</p>
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    <a href="https://2018.igem.org/Team:Hawaii/Experiments">View our experiment.</a>
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    <h1>VLP STRUCTURE AND STABILITY</h1>
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    <h2>through a combinatorial type assay</h2>
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    <p>Constructs ending at the confirmed protease cleavage site, containing variations of the capsid, with N or C-terminal purification tags, and the presence or absence of extra amino acids were amplified. Purified proteins were subjected to various VLP assembly conditions and viewed under the electron microscope.</p>
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    <a href="https://2018.igem.org/Team:Hawaii/Experiments">See our results.</a>
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<h3>What should this page contain?</h3>
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<ul>
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<li> A clear and concise description of your project.</li>
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<li>A detailed explanation of why your team chose to work on this particular project.</li>
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    <h1>DETECTING VLP ASSEMBLY</h1>
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<h3>Inspiration</h3>
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    <h2>through a fluorescent protein attachment</h2>
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<p>See how other teams have described and presented their projects: </p>
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    <p>Another construct containing our confirmed VLP forming sequence and a red fluorescent protein is designed. We attempted to express the Gag-RFP fusion protein and are continuing our efforts even after the Wiki freeze. </p>
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    <a href="https://2018.igem.org/Team:Hawaii/Experiments">See our progress.</a>
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<li><a href="https://2016.igem.org/Team:Imperial_College/Description">2016 Imperial College</a></li>
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<li><a href="https://2016.igem.org/Team:Wageningen_UR/Description">2016 Wageningen UR</a></li>
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<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> 2014 UC Davis</a></li>
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<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">2014 SYSU Software</a></li>
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We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be concise, accurate, and unambiguous in your achievements.
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<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 thought about your project and what works inspired you.</p>
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    <h1>PROJECT ABSTRACT</h1>
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    <p> &emsp;&emsp;&emsp;&emsp;&emsp; Nature has provided a remarkable system to insert genes into functional centromeres of grass genomes. Specifically, centromeric retrotransposons (CR) have the unique ability to insert themselves into the centromere by targeting a yet unidentified docking agent. We plan to adapt this system to insert genes of interest into centromeres. Centromeres are advantageous transgene targets because they lack recombination, allowing the stacking of multiple traits. Retrotransposons, or “jumping genes,” self-replicate and package their genome into self-assembling virus-like particles (VLPs), then reinsert (or “jump”) themselves into a new chromosomal location. To measure the stability of VLPs for packaging molecular cargo, we cloned the full-length gene encoding the CR gag protein and successfully generated VLPs in vitro. We also tested the efficiency of different gene constructs in forming VLPs in vitro. Electron microscopy can confirm VLP assembly, however, we plan to develop a convenient fluorescent assay to assess VLP assembly.</p>
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{{Hawaii/Footer}}

Latest revision as of 02:23, 18 October 2018

TO THE CENTROMERE

with retrotransposon VLPs

Centromere retrotransposon (CR) elements offer a natural transportation system to carry genes to the centromere. These elements utilize virus-like particles to encapsulate their genome for reverse transcription, then reintegration into the centromere. Centromeres as a transgene target allows for the accumulation of traits due to lack of recombinatorial events. We explored the virus-like particle vehicle in this system and measured the stability of the structure for packaging molecular cargo.

See our abstract.

NATURAL VLP FORMATION

using bioinformatics and a protease assay

To determine the most natural construct for VLP formation, we needed to identify the protease cut site that would allow for mature VLP formation. Initial research on aspartyl proteases allowed us to determine putative protease cleavage sites between hydrophobic amino acid residues.

To experimentally determine the protease cleavage sites, we transformed E.coli with a construct containing the gag and protease domains. Following induction, autocatalytic protease activity was observed and the resulting protein bands were sent to the Taplin Mass Spectrometry Facility. Tryptic fragments confirmed our previous putative cut site downstream of the nucleocapsid.

View our experiment.

VLP STRUCTURE AND STABILITY

through a combinatorial type assay

Constructs ending at the confirmed protease cleavage site, containing variations of the capsid, with N or C-terminal purification tags, and the presence or absence of extra amino acids were amplified. Purified proteins were subjected to various VLP assembly conditions and viewed under the electron microscope.

See our results.

DETECTING VLP ASSEMBLY

through a fluorescent protein attachment

Another construct containing our confirmed VLP forming sequence and a red fluorescent protein is designed. We attempted to express the Gag-RFP fusion protein and are continuing our efforts even after the Wiki freeze.

See our progress.

PROJECT ABSTRACT

      Nature has provided a remarkable system to insert genes into functional centromeres of grass genomes. Specifically, centromeric retrotransposons (CR) have the unique ability to insert themselves into the centromere by targeting a yet unidentified docking agent. We plan to adapt this system to insert genes of interest into centromeres. Centromeres are advantageous transgene targets because they lack recombination, allowing the stacking of multiple traits. Retrotransposons, or “jumping genes,” self-replicate and package their genome into self-assembling virus-like particles (VLPs), then reinsert (or “jump”) themselves into a new chromosomal location. To measure the stability of VLPs for packaging molecular cargo, we cloned the full-length gene encoding the CR gag protein and successfully generated VLPs in vitro. We also tested the efficiency of different gene constructs in forming VLPs in vitro. Electron microscopy can confirm VLP assembly, however, we plan to develop a convenient fluorescent assay to assess VLP assembly.