Difference between revisions of "Team:Calgary/Parts"

 
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         <br>
 
         <br>
 
         <div class="infosection">
 
         <div class="infosection">
−
            <h3 class="infosubtitle">Basic Parts</h3>
+
          <h3 class="infosubtitle">Parts Collection</h3>
 
             <br>
 
             <br>
−
           <h5><a href="http://parts.igem.org/Part:BBa_K2605000 " target="blank"><b>BBa_K2605000</b></a>- mCherry + BGH</h5>
+
           <p style="text-indent: 0px">Parts <b>BBa_K2605000 to BBa_K2605004 and BBa_K2605009</b> comprise the first
−
            <p style="text-indent: 0px">Lorem ipsum dolor sit amet consectetur adipisicing elit. Consequuntur, totam laudantium, dolor porro laboriosam
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                iteration of a novel toolkit for targeted gene integration and maintenance of expression in eukaryotic
−
                 illo tenetur velit nulla corrupti quasi non eum amet quod dolores, doloremque eius ad temporibus perferendis!
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                chassis. The collection contains chromatin-modifying elements that limit silencing of the integrated
−
                 Lorem ipsum dolor sit, amet consectetur adipisicing elit. Sit explicabo suscipit similique id expedita cum
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                 gene and minimize neighborhood effects. These elements, as well as the mCherry-BGH reporter gene with
−
                consequatur voluptatibus consectetur adipisci beatae unde, cupiditate inventore. Quis officiis quam porro
+
                 our improved CMV promoter, have restriction sites for directional cloning into part BBa_K2605002, which
−
                 a expedita non.</p>
+
                 is a Multiple Cloning Site (MCS) flanked by distinct FRT (FLP recombinase recognition target) sites.</p>
 
             <br>
 
             <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 style="text-indent: 0px">In the first iteration of our gene integration strategy,
−
             <p style="text-indent: 0px">Lorem ipsum dolor sit amet consectetur adipisicing elit. Temporibus rerum vel eius ut dolore, ab obcaecati officiis
+
                 the FRT sites are leveraged for recombinase-mediated casette exchange (RMCE).
−
                 modi porro, sunt deleniti, consequatur assumenda asperiores aliquid recusandae tenetur neque quae suscipit!
+
                 Representing an experimental second design, <b>BBa_K2605006</b> is a prototype part intended to test
−
                Lorem ipsum dolor sit amet consectetur adipisicing elit. Optio a quam iusto quo, nesciunt odit fuga, similique
+
                 our novel FLP-Beta strategy. For either RMCE or FLP-Beta, the toolkit is intended to leverage
−
                 aspernatur veritatis nemo commodi libero nobis magnam necessitatibus, quidem maiores error debitis minima.
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                 genome-integrated recombinase target sites.</p>
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                 Lorem ipsum dolor sit amet consectetur adipisicing elit. Quam ipsum consequatur, deserunt assumenda odio
+
−
                 natus. Quis, ea dolor! Voluptas dolore, facere cum illo sunt consectetur nam a soluta optio perferendis.</p>
+
 
             <br>
 
             <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 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>
  
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             <p style="text-indent: 0px">Lorem ipsum dolor sit, amet consectetur adipisicing elit. Rerum sit perferendis eum delectus odit vero saepe,
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            <img class="info-img2-div" src="https://static.igem.org/mediawiki/2018/1/14/T--Calgary--MCSNew.png">
−
                 dignissimos aspernatur et libero quisquam minima soluta a suscipit tempora dolores non aliquid ratione? Lorem
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−
                 ipsum dolor, sit amet consectetur adipisicing elit. Sunt excepturi quod, doloribus et asperiores similique
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             <p style="text-indent: 0px">The figure below shows an example of how chromatin-modifying elements and a
−
                 tempora, mollitia possimus doloremque officia deleniti eius aut dolorum fuga reiciendis adipisci esse quisquam
+
                reporter gene can be used in our system. The restriction sites for bi-directional cloning of each part
−
                 quae.
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                 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>
 
             </p>
 
             <br>
 
             <br>
−
          <h5><a href="http://parts.igem.org/Part:BBa_K2605003 " target="blank"><b>BBa_K2605003</b></a>- A2UCOE</h5>
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605003 " target="blank"><b>BBa_K2605003</b></a>- A2UCOE</h5> -->
−
          <p style="text-indent: 0px"></p>
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605003 " target="blank"><b>BBa_K2605003</b></a> - A2UCOE</b></p>
−
          <br>
+
            <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
−
          <h5><a href="http://parts.igem.org/Part:BBa_K2605004 " target="blank"><b>BBa_K2605004</b></a>- Chicken beta-globin insulator</h5>
+
                5' MfeI restriction site and 3' NsiI restriction site so it can be used within our Multiple-Cloning
−
<p style="text-indent: 0px"></p>
+
                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
 +
                beta-globin insulator</h5> -->
 +
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605004 " target="blank"><b>BBa_K2605004</b></a> - Chicken
 +
                    beta-globin insulator</b></p>
 +
            <p style="text-indent: 0px">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). </p>
 +
            <br>
 +
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605005 " target="blank"><b>BBa_K2605005</b></a> - FRT2</h5> -->
 +
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605005 " target="blank"><b>BBa_K2605005</b></a> - FRT2</b></p>
 +
            <p style="text-indent: 0px">A FLP recombinase recognition target (FRT) site.</p>
 +
            <br>
 +
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605006 " target="blank"><b>BBa_K2605006</b></a> - Hybrid
 +
                FlpO-beta resolvase</h5> -->
 +
            <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>
 
           <br>
 
           <br>
−
          <h5><a href="http://parts.igem.org/Part:BBa_K2605005 " target="blank"><b>BBa_K2605005</b></a>- FRT2</h5>
 
−
          <p style="text-indent: 0px"></p>
 
−
          <br>
 
−
          <h5><a href="http://parts.igem.org/Part:BBa_K2605006 " target="blank"><b>BBa_K2605006</b></a>- Hybrid FlpO-beta resolvase</h5>
 
−
<p style="text-indent: 0px"></p>
 
−
          <br>
 
−
         
 
 
             <h3 class="infosubtitle">Composite Parts</h3>
 
             <h3 class="infosubtitle">Composite Parts</h3>
 
             <br>
 
             <br>
−
        <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>
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605007 " target="blank"><b>BBa_K2605007</b></a> - CMV with 5'
−
          <p style="text-indent: 0px"></p>
+
                SalI restriction site + monomeric RFP optimized for bacteria</h5> -->
−
          <br>
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605007 " target="blank"><b>BBa_K2605007</b></a> - CMV with
−
   
+
                    5'
−
          <h5><a href="http://parts.igem.org/Part:BBa_K2605008 " target="blank"><b>BBa_K2605008</b></a>- CMV + monomeric RFP optimized for bacteria</h5>
+
                    SalI restriction site + monomeric RFP optimized for bacteria</b></p>
−
<p style="text-indent: 0px"></p>
+
            <p style="text-indent: 0px">Part used to characterize and validate the improvement of part BBa_K2605001.
−
          <br>
+
                Read more about its characterization <a href="https://2018.igem.org/Team:Calgary/Improve">here</a>. </p>
−
          <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>
+
            <br>
−
          <p style="text-indent: 0px"></p>
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605008 " target="blank"><b>BBa_K2605008</b></a> - CMV +
−
          <br>
+
                monomeric RFP optimized for bacteria</h5> -->
−
          <h5><a href="http://parts.igem.org/Part:BBa_K26050010 " target="blank"><b>BBa_K26050010</b></a>- CMV + mCherry + BGH</h5>
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605008 " target="blank"><b>BBa_K2605008</b></a> - CMV +
−
<p style="text-indent: 0px"></p>
+
                    monomeric RFP optimized for bacteria</b></p>
−
          <br>
+
            <p style="text-indent: 0px">Part used to characterize and validate the improvement of part BBa_K2605001.
−
          <br>
+
                Read more about its characterization <a href="https://2018.igem.org/Team:Calgary/Improve">here</a>. </p>
−
          <h3 class="infosubtitle">Parts Collection</h3>
+
            <br>
−
          <br>
+
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605009 " target="blank"><b>BBa_K2605009</b></a> - CMV with 5'
−
          <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. The MCS carries sites for an arbitrary construct to be directionally cloned. </p>
+
                SalI restriction site + mCherry + BGH</h5> -->
−
<br>
+
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605009 " target="blank"><b>BBa_K2605009</b></a> - CMV with
−
<<p style="text-indent: 0px">�In the first iteration of our gene integration strategy,
+
                    5'
−
          the FRT sites are leveraged for recombinase-mediated casette exchange (RMCE).
+
                    SalI restriction site + mCherry + BGH</b></p>
−
          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>
+
            <p style="text-indent: 0px">Composite part of BBa_K2605001 (promoter) fused to BBa_K2605000
−
<br>
+
                (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).</p>
 +
            <br>
 +
            <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605010 " target="blank"><b>BBa_K2605010</b></a> - CMV +
 +
                mCherry + BGH</h5> -->
 +
            <p><b><a href="http://parts.igem.org/Part:BBa_K2605010 " target="blank"><b>BBa_K2605010</b></a> - CMV +
 +
                    mCherry + BGH</b></p>
 +
            <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>
 +
            <br>
 +
         
 +
           
 
         </div>
 
         </div>
 
     </div>
 
     </div>

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.