Difference between revisions of "Team:Ruperto Carola/Parts"

 
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     <figcaption class="figure-caption">A caption for the above image.</figcaption>
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      <h1>Parts</h1>
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<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/>
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<div class="column full_size">
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<h1>Parts</h1>
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<p>Each team will make new parts during iGEM and will add them to the Registry of Standard Biological Parts. The iGEM 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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</div>
 
</div>
 +
<p>
 +
          During our journey of making our yeasts fantastic, we built many blocks which we wish to make reusable to further iGEM
 +
teams. We have packaged our main system for directed evolution, OrthoRep, with all the means to evolve your own proteins of interest.
 +
Furthermore, we provide a set of parts for engineering modular, orthogonal GPCRs in yeast.
 +
All in all, we provide a toolbox of parts containing:
  
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</p>
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<div class="highlight decoration_background">
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<ul>
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<h3>Note</h3>
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<li>The error-prone DNA polymerase from <i>K. lactis</i> used for OrthoRep (TP-DNAP1).</li>
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<p>Note that parts must be well documented on each part's <a href="http://parts.igem.org/Main_Page">Main Page on the Registry</a>. This documentation includes all of the characterization data for your parts. <b>The part's data MUST be on the part's Main Page on the Registry for your team to be eligible for medals and special prizes pertaining to parts.</b> <br><br>
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<li>An extensible integration cassette for evolving your own parts using our system.</li>
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This page serves to <i>showcase</i> the parts you have made and should include links to the Registry pages for your parts. 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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<li>A modular toolbox for building your own orthogonal GPCRs in <i>S. cerevisiae</i></li>
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</div>
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</ul>
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                <div class="col-12 col-md-3 pb-3 pb-md-0">
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<div class="highlight decoration_B_full">
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                    <img class="img-fluid mx-auto d-block pb-3" src="https://static.igem.org/mediawiki/2019/e/ef/T--Ruperto_Carola--dice1.png" alt="">
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                </div>
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<h3>Adding parts to the registry</h3>
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                <div class="col-12 col-md-9 d-flex justify-content-center align-items-center">
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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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                    <h2>Best basic: polymerase BBa_K3173000</h2>
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                </div>
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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. Documentation includes the characterization data of your parts.</p>
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            </div>
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<div class="button_link">
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            <div class="row mt-2">
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<a href="http://parts.igem.org/Add_a_Part_to_the_Registry">
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                <div class="col-12">
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ADD PARTS
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                    <p class="text-justify">
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</a>
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This part (terminal protein DNAP1) represents a DNA polymerase which was extracted from the K.
 +
lactis – a yeast strain actively used in biotechnology (especially fermentation) and science [<x-ref>Bergquist2002</x-ref>]. This strain is also known to harbor and
 +
express a linear cytoplasmic yeast plasmids.
 +
</p>
 +
<p class="text-justify">
 +
This part is an element of the Orthorep system, where it is responsible for replicating the engineered
 +
plasmid with the genes of interest. We used a yeast strain harbouring the aforementioned
 +
polymerase and linear plasmid, which was developed for increased error rate of the polymerase and
 +
characterized by the group of Chang Liu <x-ref>Zhong2018</x-ref>.
 +
Since the common yeast strains employed in research (S. cerevisiae) are lacking linear cytoplasmic
 +
plasmids in normal conditions, the insertion of this polymerase, together with the engineered
 +
OrthoRep system allowed us to orthogonally mutagenize genes of interest in vivo [<x-ref>Ravikumar2018</x-ref>].
 +
</p>
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
 
</div>
 
</div>
  
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</div>
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<div class="row pb-5 justify-content-center">
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    <div class="col-10 rounded shadow">
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        <div class="card-body">
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            <img class="w-25 mr-1 float-right pl-3" src="https://static.igem.org/mediawiki/2019/1/14/T--Ruperto_Carola--img-structure_swiss.png" alt="">
 +
            <p class="text-justify">
 +
To better understand the system, we first sought to take a look at the structure of the
 +
polymerase. As for general characteristics, the polymerase consists of 995 amino acids and has a
 +
mass of around 116 kDa. A search for more structure-related information in the literature turned up
 +
blank, and even finding our protein in Uniprot left us without a structure. Thus, we had to take
 +
matters into our own hands and attempt to solve the structure of TP-DNAP1. We proceeded to build
 +
homology models of the protein using SwissModel.
 +
            </p>
 +
        </div>
 +
    </div>
 
</div>
 
</div>
  
 +
<div class="row pb-5 justify-content-center">
 +
    <div class="col-10 rounded shadow">
 +
        <div class="card-body">
 +
            <img class="w-25 float-left pr-3" src="https://static.igem.org/mediawiki/2019/2/25/T--Ruperto_Carola--img-tpdnap.png" alt="">
 +
            <p class="text-justify">
  
 +
There, we were met with a pitiful amount of homology to our protein, which reliably drove the
 +
model quality into the ground. Another difficulty arose due to the lack of homological structures for a
  
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<div class="column third_size">
+
large part of the C-terminus of our protein (first 325 AA were thus not included in the model, that
−
<div class="highlight decoration_A_full">
+
correspond to the terminal protein sequence). Now, we had two options left: solve our structure de
−
<h3>Inspiration</h3>
+
novo or crystallize it, and we sure as heck wouldn't go for the last option. Therefore, we
−
<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>
+
moved on to greener pastures and submitted our sequence to both Robetta and RaptorX. The results
 +
of the de-novo structure assembly can be seen in the images below.
 +
According to the obtained images our polymerase has 4 domains including the capping protein. Here
 +
we present only the actual polymerase domain.
  
−
<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
+
</p>
−
<ul>
+
        </div>
−
<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
+
    </div>
−
<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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−
</ul>
+
−
</div>
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</div>
 
</div>
  
  
−
<div class="clear extra_space"></div>
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<div class="row pb-5 justify-content-center">
 +
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        <div class="card-body">
 +
            <div class="row">
 +
<div class="col-12 col-md-9">
 +
                    <h2>Best composite: BBa_K3173001</h2>
 +
                    <p class="text-justify">
 +
Here we present to our best composite part – the OrthoRep integration cassette. This part is the key
 +
to speeding up the evolution of your gene of interest. As the name already implies, this cassette
 +
integrates into the OrthoRep system, where all the genes on it get rapidly mutagenized by the
 +
engineered error-prone fungal orthogonal DNA polymerase. The
 +
cassette as presented here consists of several parts, for example the sequences needed for
 +
integration into the cytoplasmic plasmid which allows the system to be fully orthogonal to the rest of
 +
the yeast genome. Further, it contains several selection markers.: For instance the ampicillin
 +
resistance gene, allowing for easy selection of the modified cassette in bacteria, as well as leu2 and
 +
ura3 genes, which make it possible to first, maintain the plasmid in yeast and secondly to select for
 +
transformed colonies via standardized auxotrophy screenings (selection on drop-out media).</p>
 +
                </div>
 +
                <div class="col-12 col-md-3 pb-3 pb-md-0">
 +
                    <img class="img-fluid mx-auto d-block" src="https://static.igem.org/mediawiki/2019/9/94/T--Ruperto_Carola--img-dice2.png" alt="">
 +
                </div>
 +
             
 +
            </div>
 +
        </div>
 +
    </div>
 +
</div>
  
  
  
 +
<div class="row pb-5 justify-content-center">
 +
    <div class="col-10 rounded shadow">
 +
        <div class="card-body">
 +
            <div class="row">
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                <div class="col-12 col-md-9">
 +
                    <h2>improvement: BBa_K2407301</h2>
 +
                    <p class="text-justify">
 +
As our improvement, we present to you the Ste2 receptor integrated into the OrthoRep cassette.
 +
Ste2 originally stands for a yeast mating receptor, which is a part of the vital system for yeast sexual
 +
replication. This in its order allows for diversifying the genotype of the species.
 +
Ste2 is a transmembrane protein with a mass of around 47 kDa [<x-ref>Marsh1988</x-ref>]. In native
 +
conditions, it binds to the peptide alpha mating factor, which is 13 amino acids long.
 +
[<x-ref>Blumers1988</x-ref>] Its high sensitivity towards this small peptide as well as its wide
 +
characterization in literature makes it a suitable backbone for creating a peptide detection tool for
 +
small to middle-sized peptides. The availability of such a tool would finally close the gap that lies
 +
between the already well-established detection of large peptides and very small peptides and create
 +
new possibilities in early disease diagnostics as well as for biomonitoring and basic research.
 +
However, the mutagenesis process of such a receptor is a non-trivial task. One of the most time-
 +
consuming steps is the jump from generating a mutagenized receptor library to the actual selection
 +
of the appropriate mutants.
 +
The integration of the ste2 gene into the OrthoRep integration cassette might present an elegant
 +
solution to the described problem. As was previously described in our project proposal as
 +
well as in our best composite part page the OrthoRep system is a tool for rapid in vivo
 +
mutagenesis in yeast. The integration of the ste2 gene in the system would thus allow generating a
 +
mutant library without having to spend time and money on expensive gene block assembly and the
 +
excessive amount of additional PCRs and transformations needed to insert them into the model
 +
organisms. Further, putting a marker like GFP or his2 gene under the FUS promotor would allow for
 +
the immediate qualitative and quantitative selection of the successful mutants.
 +
The improved sequence, as well as its model, is presented below.</p>
 +
                </div>
 +
                <div class="col-12 col-md-3 pb-3 pb-md-0">
 +
                    <img class="img-fluid mx-auto d-block" src="https://static.igem.org/mediawiki/2019/2/2b/T--Ruperto_Carola--img-dice3.png" alt="">
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
 +
</div>
  
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<div class="column full_size">
 
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<h3>What information do I need to start putting my parts on the Registry?</h3>
 
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<p>The information needed to initially create a part on the Registry is:</p>
 
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<ul>
 
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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>Long Description (Longer description of what the DNA does)</li>
 
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<li>Design considerations</li>
 
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</ul>
 
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<p>
 
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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, you must also put it up on the part page. </p>
 
  
 
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<h3>Part Table </h3>
 
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<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>
 
</html>

Latest revision as of 01:56, 13 December 2019

Best basic: polymerase BBa_K3173000

This part (terminal protein DNAP1) represents a DNA polymerase which was extracted from the K. lactis – a yeast strain actively used in biotechnology (especially fermentation) and science [Bergquist2002]. This strain is also known to harbor and express a linear cytoplasmic yeast plasmids.

This part is an element of the Orthorep system, where it is responsible for replicating the engineered plasmid with the genes of interest. We used a yeast strain harbouring the aforementioned polymerase and linear plasmid, which was developed for increased error rate of the polymerase and characterized by the group of Chang Liu Zhong2018. Since the common yeast strains employed in research (S. cerevisiae) are lacking linear cytoplasmic plasmids in normal conditions, the insertion of this polymerase, together with the engineered OrthoRep system allowed us to orthogonally mutagenize genes of interest in vivo [Ravikumar2018].

To better understand the system, we first sought to take a look at the structure of the polymerase. As for general characteristics, the polymerase consists of 995 amino acids and has a mass of around 116 kDa. A search for more structure-related information in the literature turned up blank, and even finding our protein in Uniprot left us without a structure. Thus, we had to take matters into our own hands and attempt to solve the structure of TP-DNAP1. We proceeded to build homology models of the protein using SwissModel.

There, we were met with a pitiful amount of homology to our protein, which reliably drove the model quality into the ground. Another difficulty arose due to the lack of homological structures for a large part of the C-terminus of our protein (first 325 AA were thus not included in the model, that correspond to the terminal protein sequence). Now, we had two options left: solve our structure de novo or crystallize it, and we sure as heck wouldn't go for the last option. Therefore, we moved on to greener pastures and submitted our sequence to both Robetta and RaptorX. The results of the de-novo structure assembly can be seen in the images below. According to the obtained images our polymerase has 4 domains including the capping protein. Here we present only the actual polymerase domain.

Best composite: BBa_K3173001

Here we present to our best composite part – the OrthoRep integration cassette. This part is the key to speeding up the evolution of your gene of interest. As the name already implies, this cassette integrates into the OrthoRep system, where all the genes on it get rapidly mutagenized by the engineered error-prone fungal orthogonal DNA polymerase. The cassette as presented here consists of several parts, for example the sequences needed for integration into the cytoplasmic plasmid which allows the system to be fully orthogonal to the rest of the yeast genome. Further, it contains several selection markers.: For instance the ampicillin resistance gene, allowing for easy selection of the modified cassette in bacteria, as well as leu2 and ura3 genes, which make it possible to first, maintain the plasmid in yeast and secondly to select for transformed colonies via standardized auxotrophy screenings (selection on drop-out media).

improvement: BBa_K2407301

As our improvement, we present to you the Ste2 receptor integrated into the OrthoRep cassette. Ste2 originally stands for a yeast mating receptor, which is a part of the vital system for yeast sexual replication. This in its order allows for diversifying the genotype of the species. Ste2 is a transmembrane protein with a mass of around 47 kDa [Marsh1988]. In native conditions, it binds to the peptide alpha mating factor, which is 13 amino acids long. [Blumers1988] Its high sensitivity towards this small peptide as well as its wide characterization in literature makes it a suitable backbone for creating a peptide detection tool for small to middle-sized peptides. The availability of such a tool would finally close the gap that lies between the already well-established detection of large peptides and very small peptides and create new possibilities in early disease diagnostics as well as for biomonitoring and basic research. However, the mutagenesis process of such a receptor is a non-trivial task. One of the most time- consuming steps is the jump from generating a mutagenized receptor library to the actual selection of the appropriate mutants. The integration of the ste2 gene into the OrthoRep integration cassette might present an elegant solution to the described problem. As was previously described in our project proposal as well as in our best composite part page the OrthoRep system is a tool for rapid in vivo mutagenesis in yeast. The integration of the ste2 gene in the system would thus allow generating a mutant library without having to spend time and money on expensive gene block assembly and the excessive amount of additional PCRs and transformations needed to insert them into the model organisms. Further, putting a marker like GFP or his2 gene under the FUS promotor would allow for the immediate qualitative and quantitative selection of the successful mutants. The improved sequence, as well as its model, is presented below.

<groupparts>iGEM19 Ruperto_Carola</groupparts>