Difference between revisions of "Team:Calgary/Parts"

 
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         <br>
 
         <br>
 
         <div class="infosection">
 
         <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>
 
             <h3 class="infosubtitle">Basic Parts</h3>
 
             <br>
 
             <br>
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             <p style="text-indent: 0px">mCherry red fluorescent protein (RFP) codon-optimized for expression in
 
             <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
 
                 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>
+
                 improvement of the original mCherry part, <a href="http://parts.igem.org/Part:BBa_J176005" target="blank">BBa_J176005</a>.</p>
 
             <br>
 
             <br>
 
             <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605001 " target="blank"><b>BBa_K2605001</b></a>- CMV with 5'
 
             <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605001 " target="blank"><b>BBa_K2605001</b></a>- CMV with 5'
Line 50: Line 114:
 
             <p style="text-indent: 0px">Cytomegalovirus (CMV) promoter with an additional 5' SalI site, which allows
 
             <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
 
                 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
+
                 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>
 
                 learn more about its characterization and improvement <a href="https://2018.igem.org/Team:Calgary/Improve">here</a>.</p>
 
             <br>
 
             <br>
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                 integration-independent transgene expression when fused to a eukaryotic promoter. This part contains a
 
                 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
 
                 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>
+
                 Site flanked by FRT sites (BBa_K2605002). </p>
 
             <br>
 
             <br>
  
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                 of integration-independent transgene expression and prevents unrelated enhancer-promoter interactions
 
                 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
 
                 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>
+
                 so it can be used within our Multiple-Cloning Site flanked by FRT sites (BBa_K2605002). </p>
 
             <br>
 
             <br>
 
             <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605005 " target="blank"><b>BBa_K2605005</b></a> - FRT2</h5> -->
 
             <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605005 " target="blank"><b>BBa_K2605005</b></a> - FRT2</h5> -->
Line 88: Line 152:
 
             <p><b><a href="http://parts.igem.org/Part:BBa_K2605006 " target="blank"><b>BBa_K2605006</b></a> - Hybrid
 
             <p><b><a href="http://parts.igem.org/Part:BBa_K2605006 " target="blank"><b>BBa_K2605006</b></a> - Hybrid
 
                     FlpO-beta resolvase</b></p>
 
                     FlpO-beta resolvase</b></p>
−
             <p style="text-indent: 0px"></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.
−
            <br>
+
          </p>
 +
          <br>
 
             <h3 class="infosubtitle">Composite Parts</h3>
 
             <h3 class="infosubtitle">Composite Parts</h3>
 
             <br>
 
             <br>
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                     5'
 
                     5'
 
                     SalI restriction site + monomeric RFP optimized for bacteria</b></p>
 
                     SalI restriction site + monomeric RFP optimized for bacteria</b></p>
−
             <p style="text-indent: 0px">Part used to characterize and validate the improvement of part <b>BBa_K2605001</b>.
+
             <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>
 
                 Read more about its characterization <a href="https://2018.igem.org/Team:Calgary/Improve">here</a>. </p>
 
             <br>
 
             <br>
Line 104: Line 169:
 
             <p><b><a href="http://parts.igem.org/Part:BBa_K2605008 " target="blank"><b>BBa_K2605008</b></a> - CMV +
 
             <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>
 
                     monomeric RFP optimized for bacteria</b></p>
−
             <p style="text-indent: 0px">Part used to characterize and validate the improvement of part <b>BBa_K2605001</b>.
+
             <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>
 
                 Read more about its characterization <a href="https://2018.igem.org/Team:Calgary/Improve">here</a>. </p>
 
             <br>
 
             <br>
Line 112: Line 177:
 
                     5'
 
                     5'
 
                     SalI restriction site + mCherry + BGH</b></p>
 
                     SalI restriction site + mCherry + BGH</b></p>
−
             <p style="text-indent: 0px">Composite part of <b>BBa_K2605001</b> (promoter) fused to <b>BBa_K2605000</b>
+
             <p style="text-indent: 0px">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
 
                 (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
 
                 the 3' end to allow for this part to be used within our Multiple-Cloning Site flanked by FRT sites
−
                 (Bba_K2605002).</p>
+
                 (BBa).</p>
 
             <br>
 
             <br>
 
             <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605010 " target="blank"><b>BBa_K2605010</b></a> - CMV +
 
             <!-- <h5><a href="http://parts.igem.org/Part:BBa_K2605010 " target="blank"><b>BBa_K2605010</b></a> - CMV +
Line 121: Line 186:
 
             <p><b><a href="http://parts.igem.org/Part:BBa_K2605010 " target="blank"><b>BBa_K2605010</b></a> - CMV +
 
             <p><b><a href="http://parts.igem.org/Part:BBa_K2605010 " target="blank"><b>BBa_K2605010</b></a> - CMV +
 
                     mCherry + BGH</b></p>
 
                     mCherry + BGH</b></p>
−
             <p style="text-indent: 0px">Composite part of <b>BBa_K747096</b> (promoter) fused to <b>BBa_K2605000</b>
+
             <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>
 
                 (reporter gene). The part contains a SalI restriction site on the 5' end. </p>
 
             <br>
 
             <br>
−
            <br>
+
         
−
            <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>
+
−
            <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>
+
−
            <p style="text-indent: 0px">Each part in this collection (except Bba_K2605002 and Bba_K2605006) 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/2018/8/8c/T--Calgary--MCSPreStuff.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">
+
 
         </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.