Team:INSA-Lyon/Project/Modeling

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<h3>Modelling</h3>
 
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<p>We are realizing this modelling to predict how many proteins of interest can be added on each granule. Those protein will be fused with the phasins construction. <br /></p>
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<h3><font color="purple">Estimation of the number of proteins present on the surface of granules.</font></h3>
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<br /><p>We are realizing this modelling to predict how many proteins of interest can be add on each granule. This protein will be associated with the phasins, by a fusion construction. <br /></p>
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Thomas Bäckström, Jane A Brockelbank and Bernd HA Rehm, (New Zealand), published some interesting <a href="https://2010.igem.org/Team:INSA-Lyon/Project/References">data</a> about phasins and granules. <br />
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<h5><em> Estimation of the number of proteins present on the surface of granules.</em> </h5> <br />
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In their experiments , they managed to extract 3g of proteins from a liquid culture containing 10<exp>11</exp> granules by liter.<br/>
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Phasins represent almost the quasi-totality of granules in number. We can make the hypothesis that these 3g of proteins correspond to 3g of phasins. The average molecular weight of the phasins is about 70 kDa. By this way, we can now estimate the quantity of proteins in mole and then the number of proteins present in each granule, using Avogadro constant.<br/>
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<img src="http://lh6.ggpht.com/_Uc3bmii-yi0/TMdo0OkeROI/AAAAAAAAAkc/xJZA47o7D9A/Tableau1.jpg" alt="Tableau1" title="Number of Proteins" width="550" height="350"  style="
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Thomas Bäckström, Jane A Brockelbank and Bernd HA Rehm, (New Zealand), had published some interesting data about phasins and granules. <em>(“Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein”). </em><br />
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It seems that each granule presents about 2,6 10<exp>8</exp> proteins on its surface, corresponding to 330 proteins by nm².
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</p>
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In their experiments , they manage to extract 3g of proteins from a liquid culture containing 10<exp>11</exp> granules by liter. It corresponds to 0,03 ng of protein in each granule. <br />
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<p style="text-indent: 0px; text-align:center;"><em> 10<exp>11</exp>  granules ↔ 3 g of proteins <br/>
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1 granule ∶ 3.10<exp/>-11</exp> g of proteins.<br /> </em> </p>
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The phasins are the most numerous proteins of the granules. We can make the hypothesis that these 3g of proteins correspond to 3g of phasins. The average molecular weight of the phasins is about 70 kDa. We can now estimate the quantity of proteins in mole.<br />
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<p style="text-indent: 0px; text-align:center;"><em> Average weight of phasin ∶ 70 kDa. <br />
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70 000 g  ↔ 1 mole of protein. <br />
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3.10<exp>-11</exp>  g ↔ 4,29.10<exp>-16</exp>  mole </em> <br /> </p>
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Now, we can calculate the number of phasins present in each granule, using the Avogadro constant.<br /> </p>
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<br /> <p style="text-indent: 0px; text-align:center;"><em> Na=6,022.10<exp>23</exp> entities / mol <br />
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N<sub>protein</sub>=Na×4,29.10<exp>-16</exp> = 258 343 800 proteins. </em> <br /> </p>
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It seems that each granule presents about 2,6 10<exp>8</exp> proteins on its surface, corresponding to 330 proteins by nm².</p>
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<h5><em> Fusion phasin/ intein/ interest protein </em> </h5> <br />
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<h3><font color="purple">Fusion phasin/ intein/ interest protein</font></h3>
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We designed a fusion sequence that associates a phasin, an intein and a GFP. The challenge is to know the percentage of functional hybrid proteins that would be translated in our system. Indeed, our modified phasins will be in competition with the constitutive phasins, which coding sequence is present on the bacterial chromosome. In order to displace the balance, we plan to over-express the modified phasins and to associate various phasins together, to increase its affinity for the granule, as indicated in <em>Banki and Al, 2005 </em>.<br />
We designed a fusion sequence that associates a phasin, an intein and a GFP. The challenge is to know the percentage of functional hybrid proteins that would be translated in our system. Indeed, our modified phasins will be in competition with the constitutive phasins, which coding sequence is present on the bacterial chromosome. In order to displace the balance, we plan to over-express the modified phasins and to associate various phasins together, to increase its affinity for the granule, as indicated in <em>Banki and Al, 2005 </em>.<br />
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Basing on the experiments realized by this team about the purification of a Maltose Binding Protein (MBP), we can try to predict the percentage of modified phasins present in the surface of the granules. <br />
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Basing on the experiments realized by this team about the purification of a Maltose Binding Protein (MBP), we can try to predict the percentage of modified phasins present on the surface of the granules. <br />
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In a liter of culture (10<exp>11</exp> granules), they measured the presence of 36,3 mg of MBP.
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In a liter of culture (10<exp>11</exp> granules), they measured the presence of 36,3 mg of MBP.<br />
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The molecular weight of the MBP is about 45 kDa. By this way, we can estimate the quantity of MBP in mole, and then the number of phasins modified with MBP present in each granule, using the Avogadro constant .<br /> </p>
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<p style="text-indent: 0px; text-align:center;"><em> 10<exp>11</exp>  granules ↔ 0,0363 g of proteins <br />
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1 granule ∶ 3,63.10<exp>-13</exp> g of proteins.
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The molecular weight of the MBP is about 45 kDa. We can estimate now the quantity of MBP in mole.<br /> </p>
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<p style="text-indent: 0px; text-align:center;"><em> Average weight of MBP ∶ 45 kDa.<br />
 
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45 000 g  ↔ 1 mole of protein. <br />
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3,63.10 <exp>-13</exp> g ↔ 8,07.10<exp>-18</exp>  mole
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<img src="http://lh3.ggpht.com/_Uc3bmii-yi0/TMdo0O1jhRI/AAAAAAAAAkg/QkAtT5Hey0g/Tableau2.jpg" alt="Tableau1" title="Number of Proteins of Interest" width="550" height="320"  style="
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Now, we can calculate the number of phasins modified with MBP present in each granule, using the Avogadro constant.<br /> </p>
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<br /> <p style="text-indent: 0px; text-align:center;"><em> Na = 6,022.10<exp>23</exp> entities / mol<br />
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N<sub>protein</sub>=Na×8,07.10<exp>-18</exp> = 4 857 750 proteins.</em> <br /> </p>
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<p style="text-align:center;"><a href="#top">Top of Page</a></p>
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Latest revision as of 01:14, 28 October 2010





Modelling



We are realizing this modelling to predict how many proteins of interest can be added on each granule. Those protein will be fused with the phasins construction.



Estimation of the number of proteins present on the surface of granules.



Thomas Bäckström, Jane A Brockelbank and Bernd HA Rehm, (New Zealand), published some interesting data about phasins and granules.

In their experiments , they managed to extract 3g of proteins from a liquid culture containing 1011 granules by liter.
Phasins represent almost the quasi-totality of granules in number. We can make the hypothesis that these 3g of proteins correspond to 3g of phasins. The average molecular weight of the phasins is about 70 kDa. By this way, we can now estimate the quantity of proteins in mole and then the number of proteins present in each granule, using Avogadro constant.

Tableau1


It seems that each granule presents about 2,6 108 proteins on its surface, corresponding to 330 proteins by nm².



Fusion phasin/ intein/ interest protein



We designed a fusion sequence that associates a phasin, an intein and a GFP. The challenge is to know the percentage of functional hybrid proteins that would be translated in our system. Indeed, our modified phasins will be in competition with the constitutive phasins, which coding sequence is present on the bacterial chromosome. In order to displace the balance, we plan to over-express the modified phasins and to associate various phasins together, to increase its affinity for the granule, as indicated in Banki and Al, 2005 .
Basing on the experiments realized by this team about the purification of a Maltose Binding Protein (MBP), we can try to predict the percentage of modified phasins present on the surface of the granules.

In a liter of culture (1011 granules), they measured the presence of 36,3 mg of MBP.
The molecular weight of the MBP is about 45 kDa. By this way, we can estimate the quantity of MBP in mole, and then the number of phasins modified with MBP present in each granule, using the Avogadro constant .



Tableau1

It seems that each granule presents about 4,9 106 proteins on its surface, corresponding to 6 proteins by nm².

By this estimation, the ratio Modified phasins ⁄ Constitutive Phasins is about 1,8%. The promoter used in the experiments is an IPTG-induced one, the pET expression vector. It is commonly used in over-expressing protein objectives. Therefore, it will be difficult to increase this ratio.


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