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> Here 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>
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<h2>1. <a href="http://parts.igem.org/Part:BBa_K1012001">BBa_K1012001</a> Protein Phosphatase 1</h2>
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      <li>The signal sequence of the MalE gene.</li>
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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.<br><br>
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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>2.  <a href="http://parts.igem.org/Part:BBa_K1012005" >BBa_K1012005</a> <i>ompC</i>-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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This is an improved version of <a href="http://parts.igem.org/Part:BBa_R0083">BBa_R0083</a>. BBa_R0083 comprises the <i>ompC</i> promoter, containing OmpR-binding sites. To improve this brick we added a strong Ribosome Binding Site (RBS; from <a href="http://parts.igem.org/Part:BBa_B0034" target="_blank">BBa_B0034</a>) followed by Green Fluorescent Protein (<a href="http://www.parts.igem.org/Part:BBa_E0040" >BBa_E0040</a>). This was achieved by digesting BBa_R0083 with <i>Spe</i>I + <i>Pst</i>I  . The RBS from BBa_B0034 was excised with <i>Xba</i>I / <i>Pst</i>I, and ligated into the BBa_R0083. The resultant plasmid was digested with <i>Spe</i>I + <i>Pst</i>I , and was ligated with the GFP-encoding gene which had been excised from BBa_E0040 by digestion with <i>Xba</i>I / <i>Pst</i>I. The resultant plasmid, Bba_ K1012005 responds to the osmotic activation of the EnvZ by producing green fluorescence. This part has been verified to work in this way.<br><br>
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<p><em>In between the European and World Jamborees, we have successfully constructed the following BioBricks we ran out of time preparing for the European Jamboree.</em><br><br>
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<h2>3. <a href="http://parts.igem.org/Part:BBa_K1012002" >BBa_K1012002</a> The TorA (Tat-targeting) signal sequence</h2>
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          <h2>Inserts and iGEM vector</h2>
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              <img id="image-6" src="https://static.igem.org/mediawiki/2013/c/c4/IgemVectors-Dundee.jpg">
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This BioBrick contains DNA coding for the TorA (TMAO reductase) signal peptide. This part can be added at the N terminus of a protein of interest, thus targeting it for export across the bacterial cytoplasmic membrane by the twin arginine transport system (Tat) which transports folded proteins. We used this part to export pre-folded PP1 from the cytoplasm to the periplasm of <i>E. coli</i>.<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>4. <a href="http://parts.igem.org/Part:BBa_K1012004" >BBa_K1012004</a> The MalE (Sec-targeting) signal sequence</h2>
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This BioBrick contains DNA coding for the MalE signal peptide. Addition of this part to the N terminus of a desired protein will target the protein product for export by the general secretory pathway (Sec) which transports linear polypeptides. We used this part to target PP1 for export to the periplasm by the Sec machinery.
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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 cell membrane, 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 cell membrane; in the hope that it would anchor there and mop up microcystin from the cell surroundings. </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 discovered that PP1 was being transported to the periplasm of <i>E.coli</i>!</p>
 
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          <h2>MalE lodged in the inner membrane of <i>E.coli</i>!</h2>
 
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          <p>By cell fractionation and Western Blot methods, we found out that 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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Latest revision as of 14:39, 28 October 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.

1. BBa_K1012001 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.

2. BBa_K1012005 ompC-GFP reporter construct.

This is an improved version of BBa_R0083. BBa_R0083 comprises the ompC promoter, containing OmpR-binding sites. To improve this brick we added a strong Ribosome Binding Site (RBS; from BBa_B0034) followed by Green Fluorescent Protein (BBa_E0040). This was achieved by digesting BBa_R0083 with SpeI + PstI . The RBS from BBa_B0034 was excised with XbaI / PstI, and ligated into the BBa_R0083. The resultant plasmid was digested with SpeI + PstI , and was ligated with the GFP-encoding gene which had been excised from BBa_E0040 by digestion with XbaI / PstI. The resultant plasmid, Bba_ K1012005 responds to the osmotic activation of the EnvZ by producing green fluorescence. This part has been verified to work in this way.

In between the European and World Jamborees, we have successfully constructed the following BioBricks we ran out of time preparing for the European Jamboree.

3. BBa_K1012002 The TorA (Tat-targeting) signal sequence

This BioBrick contains DNA coding for the TorA (TMAO reductase) signal peptide. This part can be added at the N terminus of a protein of interest, thus targeting it for export across the bacterial cytoplasmic membrane by the twin arginine transport system (Tat) which transports folded proteins. We used this part to export pre-folded PP1 from the cytoplasm to the periplasm of E. coli.

4. BBa_K1012004 The MalE (Sec-targeting) signal sequence

This BioBrick contains DNA coding for the MalE signal peptide. Addition of this part to the N terminus of a desired protein will target the protein product for export by the general secretory pathway (Sec) which transports linear polypeptides. We used this part to target PP1 for export to the periplasm by the Sec machinery.

Retrieved from "http://2013.igem.org/Team:Dundee/Parts/Ourbiobricks"