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                                <a id="centered" href="https://2017.igem.org/Team:UIUC_Illinois/Wet_lab">Wet lab</a>                               
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    <h1>Description</h1>
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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>
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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><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.</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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    <p>Unlike traditional restriction enzyme methods, Gibson Assembly allows one to simply assemble multiple fragments of DNA in any chosen orientation without any unwanted or unnecessary DNA fragments at the junctions. Though Gibson Assembly offers a more efficient and effective cloning process, its high cost makes it pragmatically difficult to use.</p>
 
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     <p>Unfortunately, the reagents necessary for Gibson are costly and often out-of-budget for many labs who are instead forced to use less successful and more time-consuming traditional approaches. If Gibson Assembly were more widely accessible, the impact of the scientific community as a whole would be profound. Accessibility could facilitate a higher level of discovery, understanding and progress. Through the implementation of synthetic biology principles and techniques, we aim to eradicate this barrier to scientific progress by engineering a cheaper, more efficient form of Gibson Assembly.</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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<h1 id="homeH">What is<em>&nbsp;</em> Gibson Assembly?</h1>
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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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<h1 id="homeH">What are we<em>&nbsp;</em>aiming to do?</h1>
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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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<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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                                    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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<h1 id="homeH">What makes our<em>&nbsp;</em>idea significant?</h1>
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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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                                <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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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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