Difference between revisions of "Team:Calgary/Description"

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     <title>Team:Calgary - 2018.igem.org/Description</title>
 
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         <h1>OUR PROJECT</h1>
 
         <h1>OUR PROJECT</h1>
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        <hr>
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      <p>
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        <p>
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                    This year, iGEM uCalgary 2018 sought to address some of the key dilemmas affecting targeted gene integration: accuracy of desired insertions, the maximum size of recombinant DNA, and the expression of integrated DNA once it is within a host chromosome. To this extent, uCalgary developed three main systems in order to construct a cell line which could facilitate large, precise gene insertions which were then protected from transcriptional silencing and methylation upon insertion. These three systems were:
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            This year, iGEM uCalgary 2018 sought to address some of the key dilemmas affecting targeted gene
−
                </p>
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            integration: accuracy of desired insertions, the maximum size of recombinant DNA, and the expression of
−
<br>
+
            integrated DNA once it is within a host chromosome. To this extent, uCalgary developed three main systems
−
<ul>
+
            in order to construct a cell line which could facilitate large, precise gene insertions which were then
−
<li> A CRISPR/Cas9 system, to introduce a recombination target site into the host genome </li>
+
            protected from transcriptional silencing and methylation upon insertion. These three systems were:
−
<li> A FlpO/Beta resolvase system, to swap desired sequences into the genome at the recombination target site and lock them in </li>
+
        </p>
−
<li> A Chromatin Modifying Elements system, to stop transcriptional silencing and promoter methylation, as well as reduce gene crosstalk </li>
+
        <br>
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</ul>
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        <ul>
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<br>
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            <li> A CRISPR/Cas9 system, to introduce a recombination target site into the host genome </li>
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  <p>
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            <li> A FlpO/Beta resolvase system, to swap desired sequences into the genome at the recombination target
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                    Keep reading below for a breakdown of each of our three project segments!
+
                site and lock them in </li>
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                </p>
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            <li> A Chromatin Modifying Elements system, to stop transcriptional silencing and promoter methylation, as
 +
                well as reduce gene crosstalk </li>
 +
        </ul>
 +
        <br>
 +
        <p>
 +
            Keep reading below for a breakdown of each of our three project segments!
 +
        </p>
 
         <img class="info-img" src="https://static.igem.org/mediawiki/2018/7/7f/T--Calgary--SampleImage.png">
 
         <img class="info-img" src="https://static.igem.org/mediawiki/2018/7/7f/T--Calgary--SampleImage.png">
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         <br>
 
         <br>
 
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         <div class="row">
 
             <div class="col-lg-5 info">
 
             <div class="col-lg-5 info">
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                 <img style="width: 100%" src="https://static.igem.org/mediawiki/2018/7/7f/T--Calgary--SampleImage.png">
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                 <img style="width: 100%" src="https://static.igem.org/mediawiki/2018/3/31/T--Calgary--CRISPR_Description_Image.png">
 
             </div>
 
             </div>
 
             <div class="col-lg-7 info">
 
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                 </h3>
 
                 </h3>
 
                 <h5>
 
                 <h5>
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                  Inserting a landing pad into the genome to enable recombination
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                    Inserting a landing pad into the genome to enable recombination
 
                 </h5>
 
                 </h5>
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                 <p> CRISPR/Cas9 is a mechanism which induces targeted breaks into DNA, allowing the insertion of foreign DNA sequences into the break site. This method was selected for its targeted insertion properties to knock-in a Flp recognition target (FRT) site into the genome, opening the door to recombination in later steps. The FRT site can be thought of as a target, marking out a site in the genome for precision targeting by recombinase in the following stage. While the maximum knock-in size of CRISPR/Cas9 insertion is limited, the small size of our FRT was not predicted to cause any errors.
+
                 <p> CRISPR/Cas9 is a mechanism which induces targeted breaks into DNA, allowing the insertion of
 +
                    foreign DNA sequences into the break site. This method was selected for its targeted insertion
 +
                    properties to knock-in a Flp recognition target (FRT) site into the genome, opening the door to
 +
                    recombination in later steps. The FRT site can be thought of as a target, marking out a site in the
 +
                    genome for precision targeting by recombinase in the following stage. While the maximum knock-in
 +
                    size of CRISPR/Cas9 insertion is limited, the small size of our FRT was not predicted to cause any
 +
                    errors.
 
                 </p>
 
                 </p>
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                 <a href="https://2018.igem.org/Team:Calgary/CRISPR"><button type="button" class="btn btn-outline-dark">Read more</button></a>
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                 <a href="https://2018.igem.org/Team:Calgary/CRISPR"><button type="button" class="btn btn-outline-dark">Read
 +
                        more</button></a>
 
             </div>
 
             </div>
 
         </div>
 
         </div>
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                 </h5>
 
                 </h5>
 
                 <p>
 
                 <p>
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                     After CRISPR has placed the FRT site into the genome, recombination can begin. FlpO recombinase is an enzyme which causes the exchange of two pieces of DNA, as long as both contain the same FRT site. Thus, by providing recombinant DNA containing the same FRT sequence as that which was inserted into the genome using CRISPR, FlpO will integrate the recombinant DNA into the genome. Our FlpO recombination system also involves a second recombination protein known as Beta resolvase. Following the initial recombination mediated by FlpO, Beta performs a second recombination which removes many of the junk sequences contained on the recombinant plasmid, as well as its FRT site. Not only does this clean up the final insert, but it prevents the insert from being removed by FlpO down the road. If the CRISPR stage of the project is thought of as placing a target in the genome, the recombinase stage is firing DNA at the target to be integrated in.
+
                     After CRISPR has placed the FRT site into the genome, recombination can begin. FlpO recombinase is
 +
                    an enzyme which causes the exchange of two pieces of DNA, as long as both contain the same FRT
 +
                    site. Thus, by providing recombinant DNA containing the same FRT sequence as that which was
 +
                    inserted into the genome using CRISPR, FlpO will integrate the recombinant DNA into the genome. Our
 +
                    FlpO recombination system also involves a second recombination protein known as Beta resolvase.
 +
                    Following the initial recombination mediated by FlpO, Beta performs a second recombination which
 +
                    removes many of the junk sequences contained on the recombinant plasmid, as well as its FRT site.
 +
                    Not only does this clean up the final insert, but it prevents the insert from being removed by FlpO
 +
                    down the road. If the CRISPR stage of the project is thought of as placing a target in the genome,
 +
                    the recombinase stage is firing DNA at the target to be integrated in.
 
                 </p>
 
                 </p>
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                 <a href="https://2018.igem.org/Team:Calgary/FLP-Beta"><button type="button" class="btn btn-outline-dark">Read more</button></a>
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                 <a href="https://2018.igem.org/Team:Calgary/FLP-Beta"><button type="button" class="btn btn-outline-dark">Read
 +
                        more</button></a>
 
             </div>
 
             </div>
 
             <div class="col-lg-5 info">
 
             <div class="col-lg-5 info">
−
                 <img style="transform: scaleX(-1); width: 100%" src="https://static.igem.org/mediawiki/2018/7/7f/T--Calgary--SampleImage.png">
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                 <img style="transform: scaleX(-1); width: 100%" src="https://static.igem.org/mediawiki/2018/3/32/T--Calgary--FLP-Beta_Description_Image.png">
 
             </div>
 
             </div>
 
         </div>
 
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                 <img style="width: 100%" src="https://static.igem.org/mediawiki/2018/9/94/T--Calgary--CMELandingPage.png">
 
                 <img style="width: 100%" src="https://static.igem.org/mediawiki/2018/9/94/T--Calgary--CMELandingPage.png">
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                     Maintenance of integrated genes via minimization of gene silencing and neighbourhood effects
 
                     Maintenance of integrated genes via minimization of gene silencing and neighbourhood effects
 
                 </h5>
 
                 </h5>
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                 <p>Gene inserts are at risk of being rendered ineffective even after successful integration into the genome, as the spread of heterochromatin and DNA methylation can cause gene silencing. Furthermore, regulatory elements within both the insert and genome near the locus of integration may interact bidirectionally, leading to changes in gene expression known as neighbourhood effects. Chromatin modifying elements (CMEs) can help to generate an isolated, protected pocket within the genome, thereby assuring stable and sustained expression of integrated genes within eukaryotic systems.
+
                 <p>Gene inserts are at risk of being rendered ineffective even after successful integration into the
−
                 
+
                    genome, as the spread of heterochromatin and DNA methylation can cause gene silencing. Furthermore,
 +
                    regulatory elements within both the insert and genome near the locus of integration may interact
 +
                    bidirectionally, leading to changes in gene expression known as neighbourhood effects. Chromatin
 +
                    modifying elements (CMEs) can help to generate an isolated, protected pocket within the genome,
 +
                    thereby assuring stable and sustained expression of integrated genes within eukaryotic systems.
 +
 
 
                 </p>
 
                 </p>
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                 <a href="https://2018.igem.org/Team:Calgary/Chromatin_Modifying_Elements"><button type="button" class="btn btn-outline-dark">Read more</button></a>
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                 <a href="https://2018.igem.org/Team:Calgary/Chromatin_Modifying_Elements"><button type="button" class="btn btn-outline-dark">Read
 +
                        more</button></a>
 
             </div>
 
             </div>
 
         </div>
 
         </div>
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                 </h3>
 
                 </h3>
 
                 <p>
 
                 <p>
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                     Lorem ipsum dolor sit amet consectetur adipisicing elit. Assumenda nihil recusandae ea suscipit blanditiis! Ut adipisci rem,
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                     Lorem ipsum dolor sit amet consectetur adipisicing elit. Assumenda nihil recusandae ea suscipit
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                    blanditiis! Ut adipisci rem,
 
                     quisquam sed fugit libero voluptatum! Ratione vitae ipsum magnam nobis molestias, omnis hic.
 
                     quisquam sed fugit libero voluptatum! Ratione vitae ipsum magnam nobis molestias, omnis hic.
 
                 </p>
 
                 </p>
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              <a href="https://2018.igem.org/Team:Calgary/Microfluidics"><button type="button" class="btn btn-outline-dark">Read more</button></a>
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                <a href="https://2018.igem.org/Team:Calgary/Microfluidics"><button type="button" class="btn btn-outline-dark">Read
 +
                        more</button></a>
 
             </div>
 
             </div>
 
             <div class="col-lg-6 info">
 
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                     Software
 
                     Software
 
                 </h3>
 
                 </h3>
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                 <p>Each year, iGEM teams develop software in conjunction with their research.  
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                 <p>Each year, iGEM teams develop software in conjunction with their research.
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However, it is difficult to efficiently access these tools due to the sheer volume of wiki content.
+
                    However, it is difficult to efficiently access these tools due to the sheer volume of wiki content.
−
Thus, we created an online database called SARA, the Software Aggregating Research Assistant,  
+
                    Thus, we created an online database called SARA, the Software Aggregating Research Assistant,
−
which organizes software tools created by iGEM teams and allows for the simplified searching.
+
                    which organizes software tools created by iGEM teams and allows for the simplified searching.
−
SARA also provides the opportunity for old software to be updated to stay current,  
+
                    SARA also provides the opportunity for old software to be updated to stay current,
−
and decreases the likelihood that teams will create redundant software.  
+
                    and decreases the likelihood that teams will create redundant software.
 
                 </p>
 
                 </p>
−
              <a href="https://2018.igem.org/Team:Calgary/Software"><button type="button" class="btn btn-outline-dark">Read more</button></a>
+
                <a href="https://2018.igem.org/Team:Calgary/Software"><button type="button" class="btn btn-outline-dark">Read
 +
                        more</button></a>
 
             </div>
 
             </div>
 
         </div>
 
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Revision as of 08:16, 17 October 2018

Team:Calgary - 2018.igem.org/Description

OUR PROJECT

This year, iGEM uCalgary 2018 sought to address some of the key dilemmas affecting targeted gene integration: accuracy of desired insertions, the maximum size of recombinant DNA, and the expression of integrated DNA once it is within a host chromosome. To this extent, uCalgary developed three main systems in order to construct a cell line which could facilitate large, precise gene insertions which were then protected from transcriptional silencing and methylation upon insertion. These three systems were:


  • A CRISPR/Cas9 system, to introduce a recombination target site into the host genome
  • A FlpO/Beta resolvase system, to swap desired sequences into the genome at the recombination target site and lock them in
  • A Chromatin Modifying Elements system, to stop transcriptional silencing and promoter methylation, as well as reduce gene crosstalk

Keep reading below for a breakdown of each of our three project segments!


CRISPR

Inserting a landing pad into the genome to enable recombination

CRISPR/Cas9 is a mechanism which induces targeted breaks into DNA, allowing the insertion of foreign DNA sequences into the break site. This method was selected for its targeted insertion properties to knock-in a Flp recognition target (FRT) site into the genome, opening the door to recombination in later steps. The FRT site can be thought of as a target, marking out a site in the genome for precision targeting by recombinase in the following stage. While the maximum knock-in size of CRISPR/Cas9 insertion is limited, the small size of our FRT was not predicted to cause any errors.

FLP Recombinase-Beta Resolvase

Integrating our desired genes at the landing pad

After CRISPR has placed the FRT site into the genome, recombination can begin. FlpO recombinase is an enzyme which causes the exchange of two pieces of DNA, as long as both contain the same FRT site. Thus, by providing recombinant DNA containing the same FRT sequence as that which was inserted into the genome using CRISPR, FlpO will integrate the recombinant DNA into the genome. Our FlpO recombination system also involves a second recombination protein known as Beta resolvase. Following the initial recombination mediated by FlpO, Beta performs a second recombination which removes many of the junk sequences contained on the recombinant plasmid, as well as its FRT site. Not only does this clean up the final insert, but it prevents the insert from being removed by FlpO down the road. If the CRISPR stage of the project is thought of as placing a target in the genome, the recombinase stage is firing DNA at the target to be integrated in.

Chromatin Modifying Elements

Maintenance of integrated genes via minimization of gene silencing and neighbourhood effects

Gene inserts are at risk of being rendered ineffective even after successful integration into the genome, as the spread of heterochromatin and DNA methylation can cause gene silencing. Furthermore, regulatory elements within both the insert and genome near the locus of integration may interact bidirectionally, leading to changes in gene expression known as neighbourhood effects. Chromatin modifying elements (CMEs) can help to generate an isolated, protected pocket within the genome, thereby assuring stable and sustained expression of integrated genes within eukaryotic systems.

Microfluidics

Lorem ipsum dolor sit amet consectetur adipisicing elit. Assumenda nihil recusandae ea suscipit blanditiis! Ut adipisci rem, quisquam sed fugit libero voluptatum! Ratione vitae ipsum magnam nobis molestias, omnis hic.

Software

Each year, iGEM teams develop software in conjunction with their research. However, it is difficult to efficiently access these tools due to the sheer volume of wiki content. Thus, we created an online database called SARA, the Software Aggregating Research Assistant, which organizes software tools created by iGEM teams and allows for the simplified searching. SARA also provides the opportunity for old software to be updated to stay current, and decreases the likelihood that teams will create redundant software.