Difference between revisions of "Team:Calgary/Parts"

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<h1>Parts</h1>
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    <title>Team:Calgary/Parts - 2018.igem.org</title>
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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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<h3>Note</h3>
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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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        <h1 style="transform: translateY(-50%)" class="infotitle">PARTS</h1>
 +
        <br>
 +
        <div class="infosection">
 +
          <h3 class="infosubtitle">Parts Collection</h3>
 +
            <br>
 +
          <p style="text-indent: 0px">Parts <b>BBa_K2605000 to BBa_K2605004 and BBa_K2605009</b> comprise the first
 +
                iteration of a novel toolkit for targeted gene integration and maintenance of expression in eukaryotic
 +
                chassis. The collection contains chromatin-modifying elements that limit silencing of the integrated
 +
                gene and minimize neighborhood effects. These elements, as well as the mCherry-BGH reporter gene with
 +
                our improved CMV promoter, have restriction sites for directional cloning into part BBa_K2605002, which
 +
                is a Multiple Cloning Site (MCS) flanked by distinct FRT (FLP recombinase recognition target) sites.</p>
 +
            <br>
 +
            <p style="text-indent: 0px">In the first iteration of our gene integration strategy,
 +
                the FRT sites are leveraged for recombinase-mediated casette exchange (RMCE).
 +
                Representing an experimental second design, <b>BBa_K2605006</b> is a prototype part intended to test
 +
                our novel FLP-Beta strategy. For either RMCE or FLP-Beta, the toolkit is intended to leverage
 +
                genome-integrated recombinase target sites.</p>
 +
            <br>
 +
          <p style="text-indent: 0px">Each part in this collection (except BBa_K2605002 and BBaK260506) is designed
 +
                with two different restriction sites on either end to allow for simple bi-directional cloning. The
 +
                entire MCS is flanked by SapI restriction sites so that the entire cloning site and FRT sites can be
 +
                transferred to different plasmids. AflII restriction sites flank the MCS interior of the FRT sites so
 +
                that only the MCS can be transferred to different plasmids. A schematic of our MCS (BBa_K2605002) can
 +
                be seen below.</p>
  
 +
            <img class="info-img2-div" src="https://static.igem.org/mediawiki/2018/1/14/T--Calgary--MCSNew.png">
  
 +
            <p style="text-indent: 0px">The figure below shows an example of how chromatin-modifying elements and a
 +
                reporter gene can be used in our system. The restriction sites for bi-directional cloning of each part
 +
                is shown.</p>
 +
            <img class="info-img2-div" src="https://static.igem.org/mediawiki/2018/8/8c/T--Calgary--MCSWithParts.png">
 +
          <p style="text-indent: 0px"><b>Our Parts Collection:</b></p>
 +
            <table>
 +
                <tr>
 +
                    <th>Part</th>
 +
                    <th>Name</th>
 +
                </tr>
 +
                <tr>
 +
                    <td><a href=" http://parts.igem.org/Part:BBa_K2605000" target="blank">BBa_K2605000</a></td>
 +
                    <td>mCherry + BGH</td>
 +
                </tr>
 +
                <tr>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2605001 " target="blank">BBa_K2605001</a></td>
 +
                    <td>CMV with 5' SalI restriction site</td>
 +
                </tr>
 +
                <tr>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2605002 " target="blank">BBa_K2605002</a></td>
 +
                    <td>Multiple cloning site with flanking FRT sites</td>
 +
                </tr>
 +
                <tr>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2605003 " target="blank">BBa_K2605003</a></td>
 +
                    <td>A2UCOE</td>
 +
                </tr>
 +
                <tr>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2605004" target="blank">BBa_K2605004</a></td>
 +
                    <td>Chicken beta-globin insulator</td>
 +
                </tr>
 +
                <tr>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2605006" target="blank">BBa_K2605006</a></td>
 +
                    <td>Hybrid FlpO-beta resolvase</td>
 +
                </tr>
 +
                <tr>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2605009" target="blank">BBa_K2605009</a></td>
 +
                    <td>CMV with 5' SalI restriction site + mCherry + BGH</td>
 +
                </tr>
 +
            </table>
 +
            <br>
 +
            <h3 class="infosubtitle">Basic Parts</h3>
 +
            <br>
 +
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605000 " target="blank"><b>BBa_K2605000</b></a>- mCherry +
 +
                BGH</h5> -->
 +
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605000 " target="blank"><b>BBa_K2605000</b></a> - mCherry +
 +
                    BGH</b></p>
 +
            <p style="text-indent: 0px">mCherry red fluorescent protein (RFP) codon-optimized for expression in
 +
                human/mammalian cells with a BGH terminator attached. The addition of BGH makes this part an
 +
                improvement of the original mCherry part, <a href="http://parts.igem.org/Part:BBa_J176005" target="blank">BBa_J176005</a>.</p>
 +
            <br>
 +
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605001 " target="blank"><b>BBa_K2605001</b></a>- CMV with 5'
 +
                SalI restriction site</h5> -->
 +
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605001 " target="blank"><b>BBa_K2605001</b></a> - CMV with
 +
                    5'
 +
                    SalI restriction site</b></p>
 +
            <p style="text-indent: 0px">Cytomegalovirus (CMV) promoter with an additional 5' SalI site, which allows
 +
                this promoter to be used within our Multiple-Cloning Site flanked by FRT sites. This is an improvement
 +
                of part <a href="http://parts.igem.org/Part:BBa_K747096" target="blank">BBa_K747096</a> and you can
 +
                learn more about its characterization and improvement <a href="https://2018.igem.org/Team:Calgary/Improve">here</a>.</p>
 +
            <br>
 +
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605002 " target="blank"><b>BBa_K2605002</b></a>- Multiple
 +
                cloning site with flanking FRT sites</h5> -->
 +
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605002 " target="blank"><b>BBa_K2605002</b></a> - Multiple
 +
                    cloning site with flanking FRT sites</b></p>
 +
            <p style="text-indent: 0px">Multiple Cloning Site (MCS) flanked by distinct FRT (FLP recombinase
 +
                recognition target) sites. The MCS carries sites for an arbitrary construct to be directionally cloned.
 +
                The MCS was designed for our first iteration of targeted gene integration, which involved
 +
                recombinase-mediated casette exchange (RMCE), thus it was flanked by FRT sites.
 +
            </p>
 +
            <br>
 +
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605003 " target="blank"><b>BBa_K2605003</b></a>- A2UCOE</h5> -->
 +
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605003 " target="blank"><b>BBa_K2605003</b></a> - A2UCOE</b></p>
 +
            <p style="text-indent: 0px">A methylation-free CpG island that provides stable and localization of
 +
                integration-independent transgene expression when fused to a eukaryotic promoter. This part contains a
 +
                5' MfeI restriction site and 3' NsiI restriction site so it can be used within our Multiple-Cloning
 +
                Site flanked by FRT sites (BBa_K2605002). </p>
 +
            <br>
  
−
 
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605004 " target="blank"><b>BBa_K2605004</b></a> - Chicken
−
<div class="column two_thirds_size">
+
                beta-globin insulator</h5> -->
−
<div class="highlight decoration_B_full">
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605004 " target="blank"><b>BBa_K2605004</b></a> - Chicken
−
 
+
                    beta-globin insulator</b></p>
−
<h3>Adding parts to the registry</h3>
+
            <p style="text-indent: 0px">DNAse‐I hypersensitive CpG‐rich structure that provides stable and localization
−
<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>
+
                of integration-independent transgene expression and prevents unrelated enhancer-promoter interactions
−
 
+
                when flanking a transgene. This part contains a 5' BmtI restriction site and 3' BamHI restriction site
−
<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>
+
                so it can be used within our Multiple-Cloning Site flanked by FRT sites (BBa_K2605002). </p>
−
<div class="button_link">
+
            <br>
−
<a href="http://parts.igem.org/Add_a_Part_to_the_Registry">
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605005 " target="blank"><b>BBa_K2605005</b></a> - FRT2</h5> -->
−
ADD PARTS
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605005 " target="blank"><b>BBa_K2605005</b></a> - FRT2</b></p>
−
</a>
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            <p style="text-indent: 0px">A FLP recombinase recognition target (FRT) site.</p>
−
</div>
+
            <br>
−
 
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605006 " target="blank"><b>BBa_K2605006</b></a> - Hybrid
−
</div>
+
                FlpO-beta resolvase</h5> -->
−
</div>
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605006 " target="blank"><b>BBa_K2605006</b></a> - Hybrid
−
 
+
                    FlpO-beta resolvase</b></p>
−
 
+
            <p style="text-indent: 0px">This part represents a plug-and-play construct to implement the FLP-Beta strategy in a eukaryotic chassis. The coding sequence for FLPo must be added to the same vector or co-delivered with a second. This construct is intended to be subcloned into the MCS of a mammalian expression vector, downstream of a strong promoter and upstream of a terminator. To maximize the efficiency of the system, the expression vector should include a secondary resistance marker (not Puromycin) and an origin of replication to maintain a plasmid population within the cell for as long as is required for the integration reaction to proceed to completion.
−
 
+
          </p>
−
<div class="column third_size">
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          <br>
−
<div class="highlight decoration_A_full">
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            <h3 class="infosubtitle">Composite Parts</h3>
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<h3>Inspiration</h3>
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            <br>
−
<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>
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605007 " target="blank"><b>BBa_K2605007</b></a> - CMV with 5'
−
 
+
                SalI restriction site + monomeric RFP optimized for bacteria</h5> -->
−
<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605007 " target="blank"><b>BBa_K2605007</b></a> - CMV with
−
<ul>
+
                    5'
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
+
                    SalI restriction site + monomeric RFP optimized for bacteria</b></p>
−
<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
+
            <p style="text-indent: 0px">Part used to characterize and validate the improvement of part BBa_K2605001.
−
<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
+
                Read more about its characterization <a href="https://2018.igem.org/Team:Calgary/Improve">here</a>. </p>
−
</ul>
+
            <br>
−
</div>
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605008 " target="blank"><b>BBa_K2605008</b></a> - CMV +
−
</div>
+
                monomeric RFP optimized for bacteria</h5> -->
−
 
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605008 " target="blank"><b>BBa_K2605008</b></a> - CMV +
−
 
+
                    monomeric RFP optimized for bacteria</b></p>
−
<div class="clear extra_space"></div>
+
            <p style="text-indent: 0px">Part used to characterize and validate the improvement of part BBa_K2605001.
−
 
+
                Read more about its characterization <a href="https://2018.igem.org/Team:Calgary/Improve">here</a>. </p>
−
 
+
            <br>
−
 
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605009 " target="blank"><b>BBa_K2605009</b></a> - CMV with 5'
−
 
+
                SalI restriction site + mCherry + BGH</h5> -->
−
<div class="column full_size">
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605009 " target="blank"><b>BBa_K2605009</b></a> - CMV with
−
 
+
                    5'
−
<h3>What information do I need to start putting my parts on the Registry?</h3>
+
                    SalI restriction site + mCherry + BGH</b></p>
−
<p>The information needed to initially create a part on the Registry is:</p>
+
            <p style="text-indent: 0px">Composite part of BBa_K2605001 (promoter) fused to BBa_K2605000
−
<ul>
+
                (reporter gene). The part contains a SalI restriction site on the 5' end and a BmtI restriction site on
−
<li>Part Name</li>
+
                the 3' end to allow for this part to be used within our Multiple-Cloning Site flanked by FRT sites
−
<li>Part type</li>
+
                (BBa).</p>
−
<li>Creator</li>
+
            <br>
−
<li>Sequence</li>
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605010 " target="blank"><b>BBa_K2605010</b></a> - CMV +
−
<li>Short Description (60 characters on what the DNA does)</li>
+
                mCherry + BGH</h5> -->
−
<li>Long Description (Longer description of what the DNA does)</li>
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605010 " target="blank"><b>BBa_K2605010</b></a> - CMV +
−
<li>Design considerations</li>
+
                    mCherry + BGH</b></p>
−
</ul>
+
            <p style="text-indent: 0px">Composite part of BBa_K747096 (promoter) fused to BBa_K2605000
−
 
+
                (reporter gene). The part contains a SalI restriction site on the 5' end. </p>
−
<p>
+
            <br>
−
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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<h3>Part Table </h3>
+
−
 
+
−
<p>Please include a table of all the parts your team has made during your project on this page. Remember part characterization and measurement data must go on your team part pages on the Registry. </p>
+
−
 
+
−
</html>
+
−
<groupparts>iGEM18 Calgary</groupparts>
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Latest revision as of 02:25, 18 October 2018

Team:Calgary/Parts - 2018.igem.org

PARTS


Parts Collection


Parts BBa_K2605000 to BBa_K2605004 and BBa_K2605009 comprise the first iteration of a novel toolkit for targeted gene integration and maintenance of expression in eukaryotic chassis. The collection contains chromatin-modifying elements that limit silencing of the integrated gene and minimize neighborhood effects. These elements, as well as the mCherry-BGH reporter gene with our improved CMV promoter, have restriction sites for directional cloning into part BBa_K2605002, which is a Multiple Cloning Site (MCS) flanked by distinct FRT (FLP recombinase recognition target) sites.


In the first iteration of our gene integration strategy, the FRT sites are leveraged for recombinase-mediated casette exchange (RMCE). Representing an experimental second design, BBa_K2605006 is a prototype part intended to test our novel FLP-Beta strategy. For either RMCE or FLP-Beta, the toolkit is intended to leverage genome-integrated recombinase target sites.


Each part in this collection (except BBa_K2605002 and BBaK260506) is designed with two different restriction sites on either end to allow for simple bi-directional cloning. The entire MCS is flanked by SapI restriction sites so that the entire cloning site and FRT sites can be transferred to different plasmids. AflII restriction sites flank the MCS interior of the FRT sites so that only the MCS can be transferred to different plasmids. A schematic of our MCS (BBa_K2605002) can be seen below.

The figure below shows an example of how chromatin-modifying elements and a reporter gene can be used in our system. The restriction sites for bi-directional cloning of each part is shown.

Our Parts Collection:

Part Name
BBa_K2605000 mCherry + BGH
BBa_K2605001 CMV with 5' SalI restriction site
BBa_K2605002 Multiple cloning site with flanking FRT sites
BBa_K2605003 A2UCOE
BBa_K2605004 Chicken beta-globin insulator
BBa_K2605006 Hybrid FlpO-beta resolvase
BBa_K2605009 CMV with 5' SalI restriction site + mCherry + BGH

Basic Parts


BBa_K2605000 - mCherry + BGH

mCherry red fluorescent protein (RFP) codon-optimized for expression in human/mammalian cells with a BGH terminator attached. The addition of BGH makes this part an improvement of the original mCherry part, BBa_J176005.


BBa_K2605001 - CMV with 5' SalI restriction site

Cytomegalovirus (CMV) promoter with an additional 5' SalI site, which allows this promoter to be used within our Multiple-Cloning Site flanked by FRT sites. This is an improvement of part BBa_K747096 and you can learn more about its characterization and improvement here.


BBa_K2605002 - Multiple cloning site with flanking FRT sites

Multiple Cloning Site (MCS) flanked by distinct FRT (FLP recombinase recognition target) sites. The MCS carries sites for an arbitrary construct to be directionally cloned. The MCS was designed for our first iteration of targeted gene integration, which involved recombinase-mediated casette exchange (RMCE), thus it was flanked by FRT sites.


BBa_K2605003 - A2UCOE

A methylation-free CpG island that provides stable and localization of integration-independent transgene expression when fused to a eukaryotic promoter. This part contains a 5' MfeI restriction site and 3' NsiI restriction site so it can be used within our Multiple-Cloning Site flanked by FRT sites (BBa_K2605002).


BBa_K2605004 - Chicken beta-globin insulator

DNAse‐I hypersensitive CpG‐rich structure that provides stable and localization of integration-independent transgene expression and prevents unrelated enhancer-promoter interactions when flanking a transgene. This part contains a 5' BmtI restriction site and 3' BamHI restriction site so it can be used within our Multiple-Cloning Site flanked by FRT sites (BBa_K2605002).


BBa_K2605005 - FRT2

A FLP recombinase recognition target (FRT) site.


BBa_K2605006 - Hybrid FlpO-beta resolvase

This part represents a plug-and-play construct to implement the FLP-Beta strategy in a eukaryotic chassis. The coding sequence for FLPo must be added to the same vector or co-delivered with a second. This construct is intended to be subcloned into the MCS of a mammalian expression vector, downstream of a strong promoter and upstream of a terminator. To maximize the efficiency of the system, the expression vector should include a secondary resistance marker (not Puromycin) and an origin of replication to maintain a plasmid population within the cell for as long as is required for the integration reaction to proceed to completion.


Composite Parts


BBa_K2605007 - CMV with 5' SalI restriction site + monomeric RFP optimized for bacteria

Part used to characterize and validate the improvement of part BBa_K2605001. Read more about its characterization here.


BBa_K2605008 - CMV + monomeric RFP optimized for bacteria

Part used to characterize and validate the improvement of part BBa_K2605001. Read more about its characterization here.


BBa_K2605009 - CMV with 5' SalI restriction site + mCherry + BGH

Composite part of BBa_K2605001 (promoter) fused to BBa_K2605000 (reporter gene). The part contains a SalI restriction site on the 5' end and a BmtI restriction site on the 3' end to allow for this part to be used within our Multiple-Cloning Site flanked by FRT sites (BBa).


BBa_K2605010 - CMV + mCherry + BGH

Composite part of BBa_K747096 (promoter) fused to BBa_K2605000 (reporter gene). The part contains a SalI restriction site on the 5' end.