Team:Dundee/Parts/Ourbiobricks

From 2013.igem.org

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           <h2>Our Biobricks</h2>
           <h2>Our Biobricks</h2>
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           <h2>What are our Biobricks?</h2>
           <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.<br><br> Here they are: </p>
           <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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<li>The signal sequence of the MalE gene.</li>
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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 PrsA gene.</li>
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<li>The signal sequence of the TorA 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 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 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 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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      <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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      <br><i>*The HA tag was used for Western Blotting.</i>
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           <h2>Inserts and iGEM vector</h2>
           <h2>Inserts and iGEM vector</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>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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            <img id="image-6" src="https://static.igem.org/mediawiki/2013/c/c4/IgemVectors-Dundee.jpg">
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<p><i>Fig.1. Diagram showing the different microcystin mop BioBricks made by the team.</i></p>
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            <p><i>Fig.1. Diagram showing the different microcystin mop BioBricks made by the team.</i></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 (found in <i>E.coli</i>)</h2>
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        <h2>The Toxi-Tweet System:</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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        <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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          <h2>SEC pathway (Found in <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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        <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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Revision as of 14:07, 15 August 2013

iGEM Dundee 2013 · ToxiMop

Dundee 2013

Our Biobricks

What are our Biobricks?

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:

  • The signal sequence of the MalE gene.
  • The signal sequence of the PrsA gene.
  • The signal sequence of the TorA gene.
  • The PP1 (protein phosphatase 1) gene with a HA tag attached immediately downstream.
  • The signal sequence of the MalE gene, with PP1-HA attached immediately downstream.
  • The signal sequence of the PrsA gene, with PP1-HA attached immediately downstream.
  • The signal sequence of the TorA gene, with PP1-HA attached immediately downstream.

*The HA tag was used for Western Blotting.

Inserts and iGEM vector

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.

Fig.1. Diagram showing the different microcystin mop BioBricks made by the team.


The Theory Behind Our Biobricks

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.

TAT pathway (found in E.coli)

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.

SEC pathway (Found in E.coli and B.subtillus)

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. 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.


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