Difference between revisions of "Team:UCLA/Collaborations"

 
(20 intermediate revisions by the same user not shown)
Line 9: Line 9:
 
<h1 style="position:relative;top:-20px;text-decoration:none;font-family: 'Nexa Light', sans-serif;color:white;" align="middle"><b>SilkyColi: Reprogramming the physical and functional properties of synthetic silks </b></h1>
 
<h1 style="position:relative;top:-20px;text-decoration:none;font-family: 'Nexa Light', sans-serif;color:white;" align="middle"><b>SilkyColi: Reprogramming the physical and functional properties of synthetic silks </b></h1>
 
</div>
 
</div>
−
 
−
<h2> Collaborations</h2>
 
  
  
  
 
<!--CONTENT-->
 
<!--CONTENT-->
−
<div class= "content_container">
+
<div class= "content_container" style="top:300px">
 
<div class= "page_content" id= "section1">
 
<div class= "page_content" id= "section1">
 
     <div class= "content_subsection" id="background">
 
     <div class= "content_subsection" id="background">
Line 22: Line 20:
 
           <p>First, we were very motivated to discuss the potential for iterative capped assembly to open the doors for other iGEM teams to explore new features of protein synthesis. To achieve this, we collaborated with the great members of the TU Eindhoven iGEM Team, for whom we developed a cloning guide for ICA.  Our entry in their guide discussing the background, process, workflow, and troubleshooting tips necessary to utilize this system.  Our collaboration has not only benefited iGEM teams who wish to learn the technique, but also helped us gain a better sense of the implications of our research toward advanced synthetic biology. <b> We envision ICA as a cloning technique that can be widely adapted not only to those using our parts collection generate novel silk genes, but for any team who wishes to rapidly and controllably assembly long repetitive genetic structures for functional use.</b> <br /> <br /></p>
 
           <p>First, we were very motivated to discuss the potential for iterative capped assembly to open the doors for other iGEM teams to explore new features of protein synthesis. To achieve this, we collaborated with the great members of the TU Eindhoven iGEM Team, for whom we developed a cloning guide for ICA.  Our entry in their guide discussing the background, process, workflow, and troubleshooting tips necessary to utilize this system.  Our collaboration has not only benefited iGEM teams who wish to learn the technique, but also helped us gain a better sense of the implications of our research toward advanced synthetic biology. <b> We envision ICA as a cloning technique that can be widely adapted not only to those using our parts collection generate novel silk genes, but for any team who wishes to rapidly and controllably assembly long repetitive genetic structures for functional use.</b> <br /> <br /></p>
  
−
<figure style= "margin: 10px; float: right;"><img src= "https://static.igem.org/mediawiki/2015/1/1d/UCLAiGEMCloningGuideExample.png" /><figcaption  style="margin: auto;width:300px;">Figure 1: Example introductory page of the ICA method on the TU Eindhoven Cloning Guide.  Guide includes details on method background, protocols and workflow needed to design assembly pieces, and FAQ/troubleshooting guidelines.</figcaption></figure>
+
</html>
−
 
+
{{Team:TU_Eindhoven/CloningGuideViewer_HTML}}
−
<p>In fact, the sequence of the spider silk monomer differs slightly from species to species. Therefore, silk produced by different species have different physical properties.  Some monomer sequences result in silks with greater strength, while other sequences result in silks with greater flexibility.  Furthermore, individual spiders can produce multiple types of silk such as dragline and flagelliform silk, each with very different properties.<sup><a href="#references">[2]</a></sup></p>
+
<html>
−
 
+
−
<p>Much research has been done on the production of spider silk from recombinant bacteria.<sup><a href="#references">[3]</a></sup>  We want to build on this work and take it a step further.  Using the wide range of spider silk monomers as our building blocks, we want to be able to create hybrid silk proteins with specified physical properties.  By mixing and matching these different monomer sequences within one polypeptide, we hope to be able to fine tune the properties of our produced silk.  If this system could be standardized, it could be exceedingly useful for quickly producing silk to fit different a variety of different needs and purposes. </p>
+
−
 
+
−
 
+
−
<h4 id="project_goals">Project Goals</h4><p>For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2).  The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup><a href="#references">[4]</a></sup>  As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p>
+
−
</p>
+
−
 
+
−
<figure style= "margin: 10px; float: left;"><img src= "https://static.igem.org/mediawiki/2014/a/ae/CustomizingSilk2.png" /><figcaption  style="margin: auto;">Fig 2: Variations of the MaSp1 amino acid sequences between different spider species <sup><a href="#references">[6]</a></sup></figcaption></figure>
+
−
 
+
−
    </div>
+
−
<br/><br/>
+
−
<br/><br/>
+
−
<br/><br/>
+
−
<br/><br/>
+
−
<br/><br/>
+
−
<br/>
+
−
 
+
−
    <div class= "content_subsection" id="ica">
+
−
          <h2> Iterative Capped Assembly</h2>
+
−
          <p>Assembling spider silk monomers such as MaSp1 and MaSp2 together can prove to be rather difficult. Due to the repetitiveness of our silk monomers, common techniques such as Gibson assembly and direct synthesis of a full-length gene product could be ineffective and less efficient in building silk sequences. Therefore, a technique called iterative capped assembly (ICA), which allows rapid assembly of repeating monomers, would be a better option in working with our highly repetitive sequences. This technique has previously been applied in order to assemble other repetitive modules.  For example, ICA has been demonstrated to efficiently assemble very repetitive transcription activator-like effector nucleases (TALENs) up to 21 monomers long<sup><a href="#references">[5]</a></sup>.</p>
+
−
+
−
<p>ICA allows the assembly of silk monomers in different ratios and orders into a custom gene sequence of modifiable length. Gene monomers are assembled individually into a growing chain that is anchored to a solid foundation through streptavidin-biotin interaction.</p>
+
−
+
−
<p>Silk monomers for ICA were built using PCR to attach BsaI recognition site onto the MaSp sequences, as well as different end extensions in the form of 4bp overhang that is essential for ligation. BsaI is a type IIs endonuclease that cleaves outside the recognition site and therefore generates overhangs that are still part of the native silk sequence. This leaves our digested products, or building blocks, free of any remaining recognition site, which is usually formed with other types of restriction enzymes. </p>
+
−
 
+
−
 
+
−
<figure style= "margin: 10px; float: right;"><img src= "https://static.igem.org/mediawiki/2014/2/29/ICAhowitworks.png" /><figcaption  style="margin: auto; width: 300px;">Fig. 3: Schematic of ICA from Briggs, et al.<sup><a href="#references">[5]</a></sup></figcaption></figure>
+
−
+
−
<p>We have designed 3 types of overhangs that the Bsa1 enzyme can generate. These are the A overhang, (AGCA),  the B overhang (TGCA) and the C overhang (TGCT).</p>
+
−
 
+
−
 
+
−
<p>The key idea here is that monomers with the same overhang are complementary and can be ligated together. Each silk monomer was modified via pcr to have one of these overhangs at the 5’ end of the sequence and another at the 3’ end of the sequence.  One version of our silk monomer had an A overhang at the 5’ end and a B overhang at the 3’ end, which we called (5’)AB(3’).  Another version was (5’)BC(3’), and the final version was (5’)CA(3’).  We therefore end up with 3 versions of the same silk monomer.  Making these modifications for all of our different types of silk monomers potentially gives us the ability to assemble a hybrid silk gene composed of different monomers in a matter of hours.  Following restriction digestion of all our monomers  with BsaI  to create the sticky ends, each silk monomer would be able to ligate to the preceding piece due to the complementing 4bp on their ends.  For example, if we wished to ligate a MaSp 2 monomer to a (5’)BC(3’)MaSp1 monomer, we would simply add a  (5’)CA(3’) MaSp2.  With the 3 subsets of MaSP1 and MaSP2, we could eventually program gene sequence of desired physical properties with various ratios and orders of each monomer type.</p>
+
−
+
−
<p>A key aspect of ICA is that the gene to be assembled is fixed to a solid support as it is being ligated together.  Streptavidin beads act as the solid support in this case.  Conjugating our gene to the beads therefore necessitates an “initiator oligo”, a biotynilated  sequence that contains both the biobrick prefix as well as one of the A,B, or C overhangs at its 3’ end.  An advantage of fixing our growing sequence to the beads is that it allows us to remove all traces of the previous ligation in a “wash” step before adding the next silk monomer to the growing gene.</p>
+
  
−
<figure style= "margin: 10px; float: left;"><img src= "https://static.igem.org/mediawiki/2014/0/04/ICA2.PNG" /><figcaption style="margin: auto;width: 300px;">Fig. 4: Immobilization of the initiator to magnetic beads. Figure from Briggs, et al.<sup><a href="#references">[5]</a></sup></figcaption></figure>
+
<h2> First Annual Southern California iGEM Meetup </h2>
−
+
<p> The UCLA iGEM was honored to hold the first ever Southern California iGEM Meetup on Thursday, August 6th.  Team members from the University of California, San Diego (<a href="https://2015.igem.org/Team:UCSD">UCSD</a>), <a href="https://2015.igem.org/Team:LaVerne-Leos">University of La Verne and La Cañada High School</a>, and the <a href="http://biohackers.la">LA Biohackers</a> Community Lab attend our meetup at the <a href="http://mbi.ucla.edu">Molecular Biology Institute</a> at UCLA.  Here is a <a href="https://static.igem.org/mediawiki/2015/2/24/2015SoCaliGEMMeetupSlides.pdf">link</a> to the powerpoint we delivered at the meet up, and below are pictures from our event.  Teams discussed their ongoing projects and offered advice to members struggling with certain aspects of their experimental design, and discussed the pertinent issues for all iGEM teams in terms of logistics, team organization and project design. Additionally, we discussed the state of iGEM and synthetic biology as a rapidly emerging biotechnology field in the United States, especially in the context of developing scientific communications to combat rising negative publicity and misinformation toward the use and safety of genetically modified organisms. <b> Through our meet up, we hope to spur not only a collaborative experience in local teams to work together in solving synthetic biology problems, but also a space where teams can discuss  maintenance and sustained success of an iGEM team, especially in dire financial circumstances </b> We hope to increase of presence in the California community, and encourage both Northern Californian and local high school teams to attend our meet up, in an effort to sustain further project collaboration.</p>
−
<p>When dealing with repetitive sequences, regular assembly techniques would give products of various lengths due to the uncontrolled ligation among pieces. Another powerful aspect of ICA is that it increases the frequency of producing a full-length sequence that we intend to build. This is achieved by adding capping oligos that ligate to a chain where a previous monomer piece fail to attach. Any incomplete or incorrect sequence due to unsuccessful ligations would be blocked from further extending, and thus monomers that are added later would ligate to the right sequences at a higher frequency.</p>
+
−
   
+
−
<p>Once the full gene sequence has been generated, the terminator oligo containing the biobrick suffix is added to complete the assembly.  The gene can then be released from the streptavidin beads by heating and disrupting the Streptavidin biotin interaction<sup><a href="#references">[5]</a></sup>. </p>
+
−
   
+
−
<p>ICA is a proficient technique to standardize assembly of any custom gene sequence. Not only does it provide flexibility in producing custom gene sequence, it is also compatible with the iGEM biobrick system. The initiator contains the prefix sequence of a biobrick and the terminator contains the suffix sequence. These contain primer sites allow PCR amplification of the full-length constructs after the release from the beads. In addition, restriction sites within the initiator and terminator enable the insertion of the complete, assembled product into a biobrick backbone easily using Golden-Gate cloning. </p>
+
  
−
</p>
 
−
</div>
 
−
<br/><br/><br/>
 
  
 +
<figure style= "margin: 1px; float: center;"><img src= "https://static.igem.org/mediawiki/2015/0/03/UCLAiGEMMeetup1.jpeg" width="1000" /></figure>
 +
<figure style= "margin: 1px; float: center;"><img src= "https://static.igem.org/mediawiki/2015/2/27/UCLAiGEMMeetup2.jpg" width="1000" /></figure>
 +
<figure style= "margin: 1px; float: center;"><img src= "https://static.igem.org/mediawiki/2015/5/57/UCLAiGEMMeetup3.jpg" width="1000" /></figure>
 +
<figure style= "margin: 1px; float: center;"><img src= "https://static.igem.org/mediawiki/2015/b/bc/UCLAiGEMMeetup4.jpg" width="1000" /></figure>
  
−
<div class= "content_subsection" id="accomplishments">
+
<h2> iGEM Team Mentorship and Development </h2>
−
          <h2>What We've Done</h2>
+
<p> Outside of the meet up, we were honored to help assist in the development and conception of multiple iGEM teams in the local area.  Through advice and collaboration with Dr. Jennifer Tsui of the University of La Verne and David Yao, co-founder and advisor of the UCLA iGEM team, we delivered a talk at the University of La Verne to discuss our project and potential avenues for synthetic biology research at an undergraduate level.  From this seminar and from our work with Dr. Tsui, we are honored to have helped inspire and start the University of La Verne iGEM team! </p>
−
          <p>Over the course of the summer, we modified the nucleotide sequence of MaSp1 and MaSp2 so that it is both compatible with the iGEM standards as well as with our ICA assembly scheme.  We generated these sequences by ordering single stranded oligos and doing multiple rounds of pcr to assemble the full construct.  These sequences contain the biobrick prefix and suffix as well as the required Bsa1 restriction sites for the ICA/Golden Gate reaction.    Furthermore, we designed and assembled the other nucleotide sequences necessary for ICA from primers that we ordered. These sequences include the biotinylated initiator oligo, the ICA terminator oligo, as well as the capping oligos (see above for description of each).</p>
+
<figure style= "margin: 1px; float: center;"><img src= "https://static.igem.org/mediawiki/2015/a/a8/10406699_10203029549445226_7197451963913804514_n.jpg" width="1000"</figure>
−
<p>We ran some trial runs of ICA by trying to assemble only a few monomers together. We only were able to run a few trials, and unfortunately, were not able to create the full silk construct. We were able to ligate the initiator oligo to the terminator oligo, showing that the initiator oligo was successfully conjugating to the streptavidin beads and that the ligation reaction reaction worked.  Hopefully with some optimizations and troubleshooting, we can use this protocol to assemble a full length silk construct in the near future.</p>
+
<br /><br />
−
    </div>
+
<p> Additionally we are honored to begin our work in assisting and developing the projects of the Granada Hills Charter High School iGEM Team, a newly formed team organized by student Anoop Nanda and advisor Jeanette Chapleu.  We are excited about the prospects of working in conjunction with this local LAUSD high school to bring STEM Education and scientific research to the San Fernando Valley area! </p>
−
<br/><br/>
+
−
<br/><br/>
+
  
−
    <div class= "content_subsection" id="references">
+
<p> Lastly, UCLA iGEM project coordinator Fasih Ahsan was selected as a team mentor for the <a href="https://2015.igem.org/Team:TecCEM"> TecCEM iGEM team</a> through the AlumniGEM mentorship program. From this , Fasih assisting in implementing, designing, and mentoring much of the TEcCEM project, especially in the potential avenues for characterization of their BioBricks, feedback on the Jamboree presentation and project description, and in offering assistance for protocol troubleshooting. He was honored to have worked with them this summer!</p>
−
<h2>References</h2>
+
−
<p dir="ltr"><sup><span>[1] </span></sup><span>Lewis, Randolph V. "Spider silk: ancient ideas for new biomaterials."&nbsp;</span><em>Chemical reviews</em><span>&nbsp;106.9 (2006): 3762-3774.</span></p>
+
−
<p dir="ltr"><sup><span>[2] </span></sup><span>Gatesy, John, et al. "Extreme diversity, conservation, and convergence of spider silk fibroin sequences."&nbsp;</span><em>Science</em><span>&nbsp;291.5513 (2001): 2603-2605.</span></p>
+
−
<p dir="ltr"><sup><span>[3] </span></sup><span>Xia, Xiao-Xia, et al. "Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber."&nbsp;</span><em>Proceedings of the National Academy of Sciences</em><span>&nbsp;107.32 (2010): 14059-14063.</span></p>
+
−
<p dir="ltr"><sup><span>[4] </span></sup><span>Huemmerich, Daniel, et al. "Novel assembly properties of recombinant spider dragline silk proteins."&nbsp;</span><em>Current Biology</em><span>;14.22 (2004): 2070-2074.</span></p>
+
−
<p dir="ltr"><sup><span>[5] </span></sup><span>Briggs, Adrian W., et al. "Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers."</span> <em>Nucleic acids research</em><span>(2012): gks624.</span></p>
+
−
<p>[6] Teulé, Florence, et al. "A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning." <i>Nature protocols</i> 4.3 (2009): 341-355.</span></p>
+
−
<p><span>&nbsp;</span></p>
+
−
<br/>
+
−
</div>
+

Latest revision as of 21:07, 18 September 2015

iGEM UCLA





SilkyColi: Reprogramming the physical and functional properties of synthetic silks

Collaborations

Cloning Guide with TU Eindhoven

First, we were very motivated to discuss the potential for iterative capped assembly to open the doors for other iGEM teams to explore new features of protein synthesis. To achieve this, we collaborated with the great members of the TU Eindhoven iGEM Team, for whom we developed a cloning guide for ICA. Our entry in their guide discussing the background, process, workflow, and troubleshooting tips necessary to utilize this system. Our collaboration has not only benefited iGEM teams who wish to learn the technique, but also helped us gain a better sense of the implications of our research toward advanced synthetic biology. We envision ICA as a cloning technique that can be widely adapted not only to those using our parts collection generate novel silk genes, but for any team who wishes to rapidly and controllably assembly long repetitive genetic structures for functional use.

Feel free to browse through the cloning guide.

First Annual Southern California iGEM Meetup

The UCLA iGEM was honored to hold the first ever Southern California iGEM Meetup on Thursday, August 6th. Team members from the University of California, San Diego (UCSD), University of La Verne and La Cañada High School, and the LA Biohackers Community Lab attend our meetup at the Molecular Biology Institute at UCLA. Here is a link to the powerpoint we delivered at the meet up, and below are pictures from our event. Teams discussed their ongoing projects and offered advice to members struggling with certain aspects of their experimental design, and discussed the pertinent issues for all iGEM teams in terms of logistics, team organization and project design. Additionally, we discussed the state of iGEM and synthetic biology as a rapidly emerging biotechnology field in the United States, especially in the context of developing scientific communications to combat rising negative publicity and misinformation toward the use and safety of genetically modified organisms. Through our meet up, we hope to spur not only a collaborative experience in local teams to work together in solving synthetic biology problems, but also a space where teams can discuss maintenance and sustained success of an iGEM team, especially in dire financial circumstances We hope to increase of presence in the California community, and encourage both Northern Californian and local high school teams to attend our meet up, in an effort to sustain further project collaboration.

iGEM Team Mentorship and Development

Outside of the meet up, we were honored to help assist in the development and conception of multiple iGEM teams in the local area. Through advice and collaboration with Dr. Jennifer Tsui of the University of La Verne and David Yao, co-founder and advisor of the UCLA iGEM team, we delivered a talk at the University of La Verne to discuss our project and potential avenues for synthetic biology research at an undergraduate level. From this seminar and from our work with Dr. Tsui, we are honored to have helped inspire and start the University of La Verne iGEM team!



Additionally we are honored to begin our work in assisting and developing the projects of the Granada Hills Charter High School iGEM Team, a newly formed team organized by student Anoop Nanda and advisor Jeanette Chapleu. We are excited about the prospects of working in conjunction with this local LAUSD high school to bring STEM Education and scientific research to the San Fernando Valley area!

Lastly, UCLA iGEM project coordinator Fasih Ahsan was selected as a team mentor for the TecCEM iGEM team through the AlumniGEM mentorship program. From this , Fasih assisting in implementing, designing, and mentoring much of the TEcCEM project, especially in the potential avenues for characterization of their BioBricks, feedback on the Jamboree presentation and project description, and in offering assistance for protocol troubleshooting. He was honored to have worked with them this summer!