Difference between revisions of "Team:Northwestern/Parts"

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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
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<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
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    <title>Parts</title>
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        <link rel="stylesheet" type="text/css" href="https://2016.igem.org/Team:Northwestern/css/bootstrap?action=raw&ctype=text/css">
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    <h1><strong>PARTS</strong></h1>
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<h2><a href="http://parts.igem.org/Part:BBa_K2019000">BBa_K2019000</a>:</h2>
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    <h3>Histidine-tagged Wild-Type Staphylococcus Aureus CRISPR Associated Protein 9 (saCas9)</h3>
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    <p>saCas9 is a RNA guided endonuclease that has proven to be an incredibly viable genome editing tool. Compared to the more generally used streptococcus pyogenes cas9 (spCas9), saCas9 is roughly 40 kDa smaller and only sacrifices slight levels of efficiencies. The construct includes the wild type sequence of saCas9 codon optimized for Escherichia coli. Furthermore, saCas9 has a his6 tag fused to the N-terminus of the protein, which can be utilized for protein purification.</p>
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    <img src="https://static.igem.org/mediawiki/2016/c/c1/T--Northwestern--divider.svg" alt="" class="divider"/>
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    <h2><a href="http://parts.igem.org/Part:BBa_K2019001">BBa_K2019001</a>:</h2>
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    <h3>gRNA Template with Constitutively Expressed mRFP</h3>
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    <p>This construct includes a mRFP gene with a constitutive promoter (BBa_J04450) upstream of the BBa_J23119-template-sgRNA scaffolding device. The template sequence is 20 bp region flanked by BsaI at the 5’ and 3’ ends. These BsaI restriction sites allow for one to insert 20 bp guide sequences in place of the template via Golden Gate Assembly. These guide sequences will then be constitutively expressed and will guide saCas9 to target specified sequences.</p>
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    <img src="https://static.igem.org/mediawiki/2016/c/c1/T--Northwestern--divider.svg" alt="" class="divider"/>
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    <h2><a href="http://parts.igem.org/Part:BBa_K2019002">BBa_K2019002</a>:</h2>
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    <h3>gRNA Guide Sequence with Constitutively Expressed mRFP</h3>
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    <p>In the presence of saCas9, the constitutively expressed gRNA guide sequence will direct saCas9 to cut the mRFP protein, causing the cell line to lose its red fluorescence. Two overlapping primers were designed in order to insert a 20 bp guide sequence into the gRNA template construct (BBa_K2019001) via Golden Gate assembly. The guide sequence targets the 130-150bp region of the mRFP gene and has an on-target score of 74.9 and off-target score of 99.1 according to Benchling.</p>
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    <img src="https://static.igem.org/mediawiki/2016/c/c1/T--Northwestern--divider.svg" alt="" class="divider"/>
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    <h2><a href="http://parts.igem.org/Part:BBa_K2019003">BBa_K2019003</a>:</h2>
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    <h3>Periplasm Directed saCas9 via TorA Signaling Sequence (TorA-saCas9)</h3>
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    <p>TorA is a signaling sequence that utilizes the post-translational twin arginine translocation pathway to direct a fused protein to the periplasm of the cell. In this construct, the TorA sequence is fused to the N-terminus of saCas9 in an attempt to direct saCas9 to the periplasm of Escherichia coli. The saCas9 gene is from part BBa_K02019003; thus it has a histidine tag on the N-terminus of the protein.</p>
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    <img src="https://static.igem.org/mediawiki/2016/c/c1/T--Northwestern--divider.svg" alt="" class="divider"/>
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    <h2><a href="http://parts.igem.org/Part:BBa_K2019004">BBa_K2019004</a>:</h2>
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    <h3>Periplasm Directed saCas9 via DsbA Signaling Sequence (DsbA-saCas9)</h3>
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    <p>DsbA is a 57 bp signaling sequence that operates in the SRP, a cotranslational pathway that utilizes certain aspects of the sec pathway to send fused proteins to the periplasm of the cell. Similar to K2019003, DsbA is also fused onto the N-terminus of saCas9 (from part BBa_K02019003). Furthermore, expression of DsbA-saCas9 can be detected utilizing this his tag on the N-terminus of saCas9.</p>
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    <img src="https://static.igem.org/mediawiki/2016/c/c1/T--Northwestern--divider.svg" alt="" class="divider"/>
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    <h2><a href="http://parts.igem.org/Part:BBa_K2019005">BBa_K2019005</a>:</h2>
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    <h3>Periplasm Directed saCas9 via Ycdo Signaling Sequence (Ycdo-saCas9)</h3>
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    <p>The signaling sequence Ycdo utilizes the cotranslational sec pathway to direct proteins to the periplasm of the cell.  In this construct, Ycdo is fused to the N-terminus of saCas9. This Ycdo-saCas9 can be detected using a Western blot with his6 antibodies.</p>
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          <div class="col-sm-3"><img src="https://static.igem.org/mediawiki/2016/8/81/T--Northwestern--logo2.png" width="3508" height="3468" alt=""/></div>
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              <p><strong>Northwestern University</strong><br>
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                Technological Institute<br>
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                2145 Sheridan Rd<br>
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                Evanston, IL 60208</p>
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            <p><em class="fa fa-envelope" aria-hidden="true"></em> nuigem2016&lt;at&gt;gmail.com<br>
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              <em class="fa fa-twitter" aria-hidden="true"></em> <a href="https://twitter.com/igem_nu" target="_blank">@iGEM_NU</a></p>
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<h5>Note</h5>
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
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<h5>Adding parts to the registry</h5>
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<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
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<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
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<h5>What information do I need to start putting my parts on the Registry?</h5>
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<p>The information needed to initially create a part on the Registry is:</p>
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<li>Part Name</li>
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<li>Part type</li>
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<li>Creator</li>
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<li>Sequence</li>
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<li>Short Description (60 characters on what the DNA does)</li>
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<li>Design considerations</li>
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
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<h5>Inspiration</h5>
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<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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<h5>Part Table </h5>
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<groupparts>iGEM2016 Example</groupparts>
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Latest revision as of 03:24, 17 October 2016

Northwestern

 
Parts

PARTS

BBa_K2019000:

Histidine-tagged Wild-Type Staphylococcus Aureus CRISPR Associated Protein 9 (saCas9)

saCas9 is a RNA guided endonuclease that has proven to be an incredibly viable genome editing tool. Compared to the more generally used streptococcus pyogenes cas9 (spCas9), saCas9 is roughly 40 kDa smaller and only sacrifices slight levels of efficiencies. The construct includes the wild type sequence of saCas9 codon optimized for Escherichia coli. Furthermore, saCas9 has a his6 tag fused to the N-terminus of the protein, which can be utilized for protein purification.

BBa_K2019001:

gRNA Template with Constitutively Expressed mRFP

This construct includes a mRFP gene with a constitutive promoter (BBa_J04450) upstream of the BBa_J23119-template-sgRNA scaffolding device. The template sequence is 20 bp region flanked by BsaI at the 5’ and 3’ ends. These BsaI restriction sites allow for one to insert 20 bp guide sequences in place of the template via Golden Gate Assembly. These guide sequences will then be constitutively expressed and will guide saCas9 to target specified sequences.

BBa_K2019002:

gRNA Guide Sequence with Constitutively Expressed mRFP

In the presence of saCas9, the constitutively expressed gRNA guide sequence will direct saCas9 to cut the mRFP protein, causing the cell line to lose its red fluorescence. Two overlapping primers were designed in order to insert a 20 bp guide sequence into the gRNA template construct (BBa_K2019001) via Golden Gate assembly. The guide sequence targets the 130-150bp region of the mRFP gene and has an on-target score of 74.9 and off-target score of 99.1 according to Benchling.

BBa_K2019003:

Periplasm Directed saCas9 via TorA Signaling Sequence (TorA-saCas9)

TorA is a signaling sequence that utilizes the post-translational twin arginine translocation pathway to direct a fused protein to the periplasm of the cell. In this construct, the TorA sequence is fused to the N-terminus of saCas9 in an attempt to direct saCas9 to the periplasm of Escherichia coli. The saCas9 gene is from part BBa_K02019003; thus it has a histidine tag on the N-terminus of the protein.

BBa_K2019004:

Periplasm Directed saCas9 via DsbA Signaling Sequence (DsbA-saCas9)

DsbA is a 57 bp signaling sequence that operates in the SRP, a cotranslational pathway that utilizes certain aspects of the sec pathway to send fused proteins to the periplasm of the cell. Similar to K2019003, DsbA is also fused onto the N-terminus of saCas9 (from part BBa_K02019003). Furthermore, expression of DsbA-saCas9 can be detected utilizing this his tag on the N-terminus of saCas9.

BBa_K2019005:

Periplasm Directed saCas9 via Ycdo Signaling Sequence (Ycdo-saCas9)

The signaling sequence Ycdo utilizes the cotranslational sec pathway to direct proteins to the periplasm of the cell. In this construct, Ycdo is fused to the N-terminus of saCas9. This Ycdo-saCas9 can be detected using a Western blot with his6 antibodies.

Northwestern University
Technological Institute
2145 Sheridan Rd
Evanston, IL 60208

nuigem2016<at>gmail.com
@iGEM_NU