Difference between revisions of "Team:UIUC Illinois"

 
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  <p> <font size="4">T</font>he development of molecular cloning techniques was a turning point in the fields of molecular biology and genetics. It suddenly allowed scientists to isolate and study individual genes from a larger system. Molecular cloning is the process by which recombinant DNA molecules are made and transformed into a host cell, where they are then replicated. Two components are necessary for a molecular cloning reaction to occur.
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      <li>A DNA segment of interest to be replicated</li>
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      <li>A vector/plasmid backbone that contains all of the elements needed for replication/expression in the host </li><br><br>
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    <p>Traditional cloning protocols used restriction enzymes to excise the DNA of interest from its source DNA. The DNA fragment was then amplified and joined to a plasmid backbone. The plasmid is a small, circular piece of DNA that is replicated within the host, and exists separately from the host’s chromosomal or genomic DNA. Physically joining the DNA of interest to the plasmid vector ensures that the DNA of interest will be fully integrated in the recombinant plasmid and thus replicated by the host.
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    <p>Although traditional cloning methods were revolutionary, there were several caveats to the process that set the stage for an updated, more efficient version of this technique: Gibson Assembly. Gibson assembly is a method of cloning that capitalizes on the properties of 3 common microbial enzymes; 5' exonuclease, polymerase and ligase.
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        <li><p><b>5' exonuclease</b> digests the 5' end of double stranded DNA to generate 3' single-stranded overhangs. The newly generated ends feature an area of 20-40 base pairs that is homologous to two or more DNA fragments in the target plasmid. Thus, the exonuclease creates complementary "sticky ends" which will efficiently find the homologous DNA pieces and anneal. The sticky ends are similar to the ends generated when using restriction enzymes except that these sticky ends have a larger region of complementarity.</p></li>
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        <li><b>DNA polymerase</b> fills any remaining sections of single-stranded DNA after the DNA sections have annealed.</li>
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        <li><b>DNA Ligase</b> then joins the segments into one continuous DNA fragment by filling in any gaps or nicks.</li>
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<h5> Wiki template information </h5>
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<p>We have created these wiki template pages to help you get started and to help you think about how your team will be evaluated. You can find a list of all the pages tied to awards here at the <a href="https://2017.igem.org/Judging/Pages_for_Awards">Pages for awards</a> link. You must edit these pages to be evaluated for medals and awards, but ultimately the design, layout, style and all other elements of your team wiki is up to you!</p>
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<h2 id="topHeader">Welcome <br> to the 2017 UIUC iGEM Wiki!</h2>
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<h1 class="section-heading" style="font-family: 'Ubuntu', sans-serif; font-weight:bolder; font-size: 48px; color: black;"><i class="fa fa-flask" aria-hidden="true" style="color:rgb(61, 209, 223); font-size: 60px;"></i>        Homemade Gibson Assembly Recipe</h1>
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<div id="start" class="col-md-10 col-md-offset-1 fh5co-heading animate-box">
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<h1 id="homeH">What is<em>&nbsp;</em> Gibson Assembly?</h1>
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<br>
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<p id="homeP">
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Gibson assembly is a useful molecular cloning technique published in 2009 by Dr. Daniel Gibson and his colleagues(Gibson, D.G., et al. (2009) Nat. Methods 6, 343-345). This technique allows for the assembly of up to 15 different DNA fragments in a single in vitro reaction. Despite its efficient process, Gibson assembly is an expensive cloning procedure because it requires high amounts of Taq DNA ligase.
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<h5>Tips</h5>
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<h1 id="homeH">What are we<em>&nbsp;</em>aiming to do?</h1>
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<p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
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<br>
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<ul>
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<p id="homeP">
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<li>State your accomplishments! Tell people what you have achieved from the start. </li>
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Due to its ease of genetic manipulation and its high replication rate, <i>Escherichia coli</i> has developed into a useful tool for researchers to express genes of interest. We believe that we can use engineered <i>E. coli</i> to overcome the high cost of using Gibson assembly method. The gene for Phusion DNA polymerase and Taq DNA ligase, two of the three proteins needed for Gibson assembly, could each be inserted into a high expression vector backbone. </p>
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<li>Be clear about what you are doing and how you plan to do this.</li>
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<p id="homeP">These construct backbones are assembled via Gibson Assembly and transformed into <i>E. coli</i>. The <i>E. coli</i> strains containing the assembled plasmids will use the local machinery to naturally express the proteins encoded in the genes inserts. The third protein required for Gibson assembly, the T5 5’ exonuclease, will be used from the natural amounts of exonuclease produced in the <i>E. coli</i> cell.
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<li>You have a global audience! Consider the different backgrounds that your users come from.</li>
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<li>Make sure information is easy to find; nothing should be more than 3 clicks away. </li>
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<li>Avoid using very small fonts and low contrast colors; information should be easy to read. </li>
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<li>Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the <a href="https://2017.igem.org/Calendar">iGEM 2017 calendar</a> </li>
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<li>Have lots of fun! </li>
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                                <p id="homeP">
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                                    Cell lysate of the <i>E. coli</i> cells containing Phusion DNA polymerase enzyme and the cell lysate for the <i>E. coli</i> cells containing Taq DNA ligase will be added in ratios to a single tube and assemble the insert and vector into an a plasmid construct. 
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<h5>Inspiration</h5>
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<h1 id="homeH">What makes our<em>&nbsp;</em>idea significant?</h1>
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<p> You can also view other team wikis for inspiration! Here are some examples:</p>
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<p id="homeP">
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<li> <a href="https://2014.igem.org/Team:SDU-Denmark/"> 2014 SDU Denmark </a> </li>
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The current cost of performing ten Gibson assembly reactions using the NEB Gibson assembly kit is <b id="fontp">$185</b>. One Gibson assembly reaction thus costs <b id="fontp">$18.50</b>. The method we are developing will be significantly cheaper amounting to <b id="fontp">$0.16</b> for two tubes worth of cell lysate including the lysozyme and LB media, and for the Gibson assembly buffer including the NAD and 10mM NEB deoxynucleotide (dNTP) solution mix (for ten reactions) needed for the Gibson assembly buffer.
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<li> <a href="https://2014.igem.org/Team:Aalto-Helsinki">2014 Aalto-Helsinki</a> </li>
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<li> <a href="https://2014.igem.org/Team:LMU-Munich">2014 LMU-Munich</a> </li>
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<li> <a href="https://2014.igem.org/Team:Michigan"> 2014 Michigan</a></li>
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<li> <a href="https://2014.igem.org/Team:ITESM-Guadalajara">2014 ITESM-Guadalajara </a></li>
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<li> <a href="https://2014.igem.org/Team:SCU-China"> 2014 SCU-China </a></li>
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                                <p id="homeP">This year, 300 teams are participating in the iGEM competition. If every team were to submit one part and use the NEB Gibson assembly master mix, the total cost would be <b id="fontp">$5,550</b>. If our recipe was used for assembling once construct per team, the cost would be <b id="fontp">$48</b>. Our method would save the 2017 iGEM teams <b id="fontp">$5,502</b> collectively. </p><br>
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<p> You can upload your pictures and files to the iGEM 2017 server. Remember to keep all your pictures and files within your team's namespace or at least include your team's name in the file name. <br />
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When you upload, set the "Destination Filename" to <br><code>T--YourOfficialTeamName--NameOfFile.jpg</code>. (If you don't do this, someone else might upload a different file with the same "Destination Filename", and your file would be erased!)<br><br>
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Latest revision as of 03:47, 2 November 2017

Homemade Gibson Assembly Recipe




What is  Gibson Assembly?


Gibson assembly is a useful molecular cloning technique published in 2009 by Dr. Daniel Gibson and his colleagues(Gibson, D.G., et al. (2009) Nat. Methods 6, 343-345). This technique allows for the assembly of up to 15 different DNA fragments in a single in vitro reaction. Despite its efficient process, Gibson assembly is an expensive cloning procedure because it requires high amounts of Taq DNA ligase.

What are we aiming to do?


Due to its ease of genetic manipulation and its high replication rate, Escherichia coli has developed into a useful tool for researchers to express genes of interest. We believe that we can use engineered E. coli to overcome the high cost of using Gibson assembly method. The gene for Phusion DNA polymerase and Taq DNA ligase, two of the three proteins needed for Gibson assembly, could each be inserted into a high expression vector backbone.

These construct backbones are assembled via Gibson Assembly and transformed into E. coli. The E. coli strains containing the assembled plasmids will use the local machinery to naturally express the proteins encoded in the genes inserts. The third protein required for Gibson assembly, the T5 5’ exonuclease, will be used from the natural amounts of exonuclease produced in the E. coli cell.

Cell lysate of the E. coli cells containing Phusion DNA polymerase enzyme and the cell lysate for the E. coli cells containing Taq DNA ligase will be added in ratios to a single tube and assemble the insert and vector into an a plasmid construct.

What makes our idea significant?


The current cost of performing ten Gibson assembly reactions using the NEB Gibson assembly kit is $185. One Gibson assembly reaction thus costs $18.50. The method we are developing will be significantly cheaper amounting to $0.16 for two tubes worth of cell lysate including the lysozyme and LB media, and for the Gibson assembly buffer including the NAD and 10mM NEB deoxynucleotide (dNTP) solution mix (for ten reactions) needed for the Gibson assembly buffer.

This year, 300 teams are participating in the iGEM competition. If every team were to submit one part and use the NEB Gibson assembly master mix, the total cost would be $5,550. If our recipe was used for assembling once construct per team, the cost would be $48. Our method would save the 2017 iGEM teams $5,502 collectively.


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