Team:Dundee/Parts/Ourbiobricks

From 2013.igem.org

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          <h2>Our Biobricks</h2>
 
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           <h2>What are our Biobricks?</h2>
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           <h2>Biobricks</h2>
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          <p>We submitted 7 constructs to the Registry of Standard Biological Parts, related to our biological mop system for the removal of microcystin from freshwater.<br><br> They are: </p>
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<p>We have submitted two BioBricks to the Registry of Standard Biological Parts that will hopefully be of use to future teams and projects.</p><br><br>  
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      <ul style="padding-left:25px;">
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<h2>Protein Phosphatase 1</h2>
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      <li>The signal sequence of the MalE gene.</li>
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<p>Human Protein Phosphatase 1 (PP1) is a protein from the family of serine/threonine phosphatases, we have used it as a microcystin binding protein however it regulates many processes in the body therefore it may be used in many other ways.</p>  
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      <li>The signal sequence of the PrsA gene.</li>
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      <li>The signal sequence of the TorA gene.</li>
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      <li>The PP1 (protein phosphatase 1) gene with a HA tag attached immediately downstream.</li>
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      <li>The signal sequence of the MalE gene, with PP1-HA attached immediately downstream.</li>
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      <li>The signal sequence of the PrsA gene, with PP1-HA attached immediately downstream.</li>
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      <li>The signal sequence of the TorA gene, with PP1-HA attached immediately downstream.</li>
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      <br><i>*The HA tag was used for Western Blotting.</i><br><br>
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<h2>OmpC-GFP reporter construct.</h2>
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      <p>All of our inserts and the iGEM vector pSB1C3 were digested at PstI and EcoRI restriction sites and then ligated together to produce our BioBricks.</p>
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<p>This reporter gene was constructed in order to test the EnvZ receptor. It consists of three parts: The ompC promoter (which is regulated by OmpR), a ribosome binding site and the GFP gene. This will ensure that upon activation of the EnvZ receptor, cells will start expressing GFP. <a href="https://www.google.co.uk/">This part has been verified to work in this way.</a></p><br><br>
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<p><em>Due to the time constraints we have been unable to submit the three BioBricks described below however, we hope to submit those once the registry re-opens again.</em><br><br>
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<h2>The signal sequence of MalE</h2>
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          <h2>Inserts and iGEM vector</h2>
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Signal allowing for protein transport via the general secretory pathway (Sec) in <i>E. coli</i><br><br>
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              <img id="image-6" src="https://static.igem.org/mediawiki/2013/c/c4/IgemVectors-Dundee.jpg">
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<h2>The signal sequence of PrsA</h2>
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Signal allowing for protein transport by the general secretory pathway (Sec) in <i>B. subtilis</i>. This signal sequence also contains a lipobox, facilitating anchoring to the outer leaflet of the cytoplasmic membrane.<br><br>  
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            <br><br><p><i>Fig.1. Diagram showing the different microcystin mop BioBricks made by the team.</i></p>
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<h2>The signal sequence of TorA.</h2>
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Signal allowing for protein transport via the <i>E. coli</i> Twin Arginine Translocation pathway (Tat) into the periplasm.</p>
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          <h2>The Theory Behind Our Biobricks</h2>
 
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          <p>As we were looking into different methods of transportation of PP1 to the cell membrane, we considered both the Tat and Sec protein targeting pathways. </p>
 
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          <h2>Tat pathway (<i>E.coli</i>)</h2>
 
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          <p>The Tat pathway is a biological method for the transport of proteins into the periplasm, with the protein being folded before entry to the plasma membrane. The protein TorA is an example of such proteins controlled by Tat. We combined the signal sequence of the TorA gene with PP1-HA in order to transport protein phosphatase 1 to the periplasm where it may act as a mop for any microcystin which traverses the outer membrane.</p>
 
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          <h2>Sec pathway (<i>E.coli</i> and <i>B.subtillus</i>)</h2>
 
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          <p>The Sec pathway is another biological method for integrating proteins into the cell membrane, with the protein being folded as it enters the plasma membrane. MalE and PrsA are both proteins destined to form a union with the cell membrane by the Sec technique.
 
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          By each combining the signal sequences MalE and PrsA with PP1-HA separately, we had the same aim for the transport of PP1 within the cell after production, as with TorA in the Tat pathway.</p>
 
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          <h2>Success and Progress</h2>
 
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          <h2>TorA signal sequence works</h2>
 
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          <p>The combination of the signal sequence of TorA with PP1-Ha proved worth our time and effort, as when we carried out cell fractionation on cells expressing this construct, and carried out Western Blots of the fractions, we found that that PP1 was being transported to the periplasm of <i>E.coli</i></p>
 
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          <h2>MalE in <i>E.coli</i>'s inner membrane</h2>
 
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          <p>By performing cell fractionation and Western blots, we obtained data that suggets MalE signal sequence joined to PP1-HA became stuck in the inner membrane of E.coli cells, therefore wouldn’t be available to bind microcystin from the cell surroundings.</p>
 
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Revision as of 09:55, 30 September 2013

iGEM Dundee 2013 · ToxiMop

Dundee 2013

Biobricks

We have submitted two BioBricks to the Registry of Standard Biological Parts that will hopefully be of use to future teams and projects.



Protein Phosphatase 1

Human Protein Phosphatase 1 (PP1) is a protein from the family of serine/threonine phosphatases, we have used it as a microcystin binding protein however it regulates many processes in the body therefore it may be used in many other ways.

OmpC-GFP reporter construct.

This reporter gene was constructed in order to test the EnvZ receptor. It consists of three parts: The ompC promoter (which is regulated by OmpR), a ribosome binding site and the GFP gene. This will ensure that upon activation of the EnvZ receptor, cells will start expressing GFP. This part has been verified to work in this way.



Due to the time constraints we have been unable to submit the three BioBricks described below however, we hope to submit those once the registry re-opens again.

The signal sequence of MalE

Signal allowing for protein transport via the general secretory pathway (Sec) in E. coli

The signal sequence of PrsA

Signal allowing for protein transport by the general secretory pathway (Sec) in B. subtilis. This signal sequence also contains a lipobox, facilitating anchoring to the outer leaflet of the cytoplasmic membrane.

The signal sequence of TorA.

Signal allowing for protein transport via the E. coli Twin Arginine Translocation pathway (Tat) into the periplasm.


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