Team:Dundee/Parts/Ourbiobricks

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          <h2>Our Biobricks</h2>
 
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          <h2>Biobricks</h2>
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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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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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<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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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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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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          <h2>What are our 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. Here they are: </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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<li>The signal sequence of the MalE gene.</li>
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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>
 
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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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        <h2>The Toxi-Tweet System:</h2>
 
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        <p>We considered different limiting factors of our mop bacteria.  The factor discussed in this section is the maximum number of PP1 which can fit either on the surface of B.subtilis, or in the periplasm of E.coli.  We considered the volumes of the bacteria and PP1 and used a cube approximation that took into account volume which was wasted, in packing, by the spherical shape of the protein. For this model we assumed there were no other surface proteins and protein production was not limited by any factors.</p><br>
 
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        <p>Calculations show the maximum number of PP1 which can fit on the surface of B.subtilis is between 60 000 -70 000. From the average we can calculate that the number of bacterial mops required to clean a toxic level of microcystin in a litre of water is 1.40x1010.</p><br>
 
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        <p>In E.coli, PP1 which would bind microcystin is free-flowing in the periplasm. The volume of the periplasm is much greater than the surface of B.subtilis. Therefore E.coli has the capacitive potential to be a more efficient mop. The maximum number of PP1 which can be packed into the periplasm is between 150 000 -200 000. Consequently, less bacterial mops are required to clean the same level of microcystin: 0.52x1010.</p><br>
 
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        <p>When we have accurate numbers from the biology team on how many PP1 are attached to the surface or in the periplasm for B.subtilis and E.coli respectively, we can compare these numbers and compute the efficiency of our PP1 expressing bacteria.</p><br>
 
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        <h2>Progress and Future Plans </h2><br>
 
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        <p>An Ordinary Differential Equation (ODE) uses a function f(t) to describe how the output changes as a result of changing the input dx(t)/dt. For example how PP1 concentration changes with time in a single cell. In order to model transcription and translation of PP1 we used a system of ODEs , which is more than one ODE where the outputs are coupled.</p><br>
 
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        <p>We used law of mass action to obtain a system of ODEs to describe the production of mRNA to PP1. mRNA and PP1 are coupled in the sense we need mRNA before we can produce any PP1. Also, the mRNA is not used up. We also took into consideration the degradation rates of mRNA and PP1 which are denoted as .</p><br>
 
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        <li>k1 – rate mRNA production - 4.98x10-9</li>
 
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        <li>kd1 – rate mRNA degradation – 1x10-2</li>
 
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        <li>k2 – rate PP1 production – 4x10-2</li>
 
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        <li>kd2 – rate PP1 degradation – 4x10-4</li>
 
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        <br> <p><i><b>Figure 1.</b> How mRNA and PP1 are produced over 20 minute cell division time. Note scaling on PP1 compared to mRNA.</i></p><br>
 
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          <p><br><i>Figure 2. A steady state is when the quantities describing a system are independent of time – they reach an equilibrium i.e dx/dt = 0. The steady state for (mRNA, PP1) is (0.04, 0.04) corresponding to a non-dimensionalised system. This plot demonstrates that during a 20 minute cell division period mRNA reaches the steady state but PP1 does not.</i></p><br>
 
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        <br><p><i>Figure 3. This plot shows that given a time longer than cell division time both the mRNA and PP1 eventually reach their steady states.</i></p><br>
 
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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"