Difference between revisions of "Part:BBa K245129"
 
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===Usage and Biology===
 
===Usage and Biology===
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<b>Group: CU 2019</b>
 
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<h1><b>In silico protein modeling</b></h1>
 
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<p>Using Expasy ProtParam tool*, theoretical protein extinction coefficient has been identified in addition to several other factors that predict the protein stability, pI and halophilicity.</p>
 
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https://2019.igem.org/wiki/images/a/ac/T--CU--Table1.png
 
  
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After which we aimed to identify the protein 3D structure and which ligand it binds by using molecular dynamics protein function prediction tool which is I-tasser.
 
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Id of the work submitted is: S494196
 
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<h2>Predicted Model with the least free energy</h2>
 
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https://2019.igem.org/wiki/images/0/05/T--CU--1.gif
 
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<h1>Characterisation by CU 2019 team</h1>
  
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The biological process identified in the model in response to metal ion, through the ligand binding site prediction, we were able to predict that CutA could bind to sodium ion;Gene ontology predicted in the model belonged to copper binding.  
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<b>In silico protein modeling</b>
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<p>First step in characterising CutA1(BBa_k245129), we used the Expasy ProtParam tool, where the theoretical protein extinction coefficient has been identified in addition to several other factors[Table1]. </p>
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<table style="width:95%">
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  <tr>
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    <th>Extiction Coeficcient</th>
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    <th>Gravy</th>
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    <th>Molecular Weight</th>
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    <th>PI</th>
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    <th>Instability Index</th>
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    <th> Aliphatic Index</th>
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    <th>kDa</th>
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  </tr>
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  <tr>
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      <td>20690</td>
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      <td>-0.037</td>
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      <td>17791.29</td>
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    <td>5.40</td>
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    <td>40.03
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"unstable"
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</td>
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    <td>85.84</td>
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    <td>12.321</td>
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  </tr>
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</table>
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<p> Then we identified the protein 3D structure[fig.1] and the ligand it binds to[fig.2] using I-tasser.
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I-tasser was used to view the folding structure and predict its affinity to bind  metal ligands in addition, to confirm the molecular function.
 +
where the model generated was closely related to Crystal structure of possible CutA1 divalent ion tolerance protein from Cryptosporidium parvum Iowa II, with TM-Score 0.95, and sequence coverage 0.9991. </p>
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<div style="width:image width 300px; font-size:90%; text-align:center;"><img src="https://2019.igem.org/wiki/images/9/92/T--CU--CutA_model1.png" alt="" width="300px" height="300px" ;" /><br>Fig1.Predicted Model with the least free energy</br></div>
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<div style="width:image width 300px; font-size:90%; text-align:center;"><img src="https://2019.igem.org/wiki/images/f/f6/T--CU--ligand.png" alt="" width="300px" height="300px" ;" /><br>Fig.2 Model showing protein binding to sodium ion with c-score: 0.1</br></div>
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<p>The biological process identified in the model in response to metal ion, through the ligand binding site prediction,predicts that CutA1 could bind to sodium ion;Gene ontology predicted in the model belonged to copper binding.</p>
 
              
 
              
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<h3>Modeling the protein binding to sodium ion with c-score: 0.1</h3>
 
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https://2019.igem.org/wiki/images/e/e1/T--CU--2.gif
 
  
 
<h1><b>CutA production using Linear expression cell-free kit</b></h1>  
 
<h1><b>CutA production using Linear expression cell-free kit</b></h1>  
 
The protein produced using Ls70 linear expression cell-free kit, under the control of constitutive family member promotor (J23102 ) and strong RBS ( B0034).
 
The protein produced using Ls70 linear expression cell-free kit, under the control of constitutive family member promotor (J23102 ) and strong RBS ( B0034).
−
We identified protein concentration by measuring the absorbance at 280nm, concentration is calculated by beer’s law using the previously identified theoretical extinction coefficient.   
+
We identified protein concentration by measuring the absorbance at 280nm, concentration was calculated by beer’s law (A = εbc) using the theoretical extinction coefficient shown in Table1.   
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We plotted the time of reaction vs the concentration of the protein produced to characterize the part production using the linear cell-free kit.  
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We then plotted the time of reaction vs the concentration of the protein produced.
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It was found that the highest production was after 14hrs[fig.3]
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<div style="width:image width 350px; font-size:90%;"><img src="https://2019.igem.org/wiki/images/thumb/a/a3/T--CU--CutA1.png/800px-T--CU--CutA1.png" alt="" width="300px" height="300px" ;" /><br>Fig3.Showing CutA1 conc at different time intervals</br></div>
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https://2019.igem.org/wiki/images/thumb/2/29/T--CU--cutA1.png/320px-T--CU--cutA1.png
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<h1><b>Testing the binding affinities of CutA1.</h1></b>
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We made a comparison between the ability of CutA1 protein to reduce both the TDS of Sodium salt and the Metal salt that it originally binds to; which is in this case Copper. So we measured the TDS before and after adding CutA1 to Copper Sulfate solution and Sodium Chloride Solution , We have found that CutA1 reduced the TDS of NaCl solution by approximately 900ppm [fig. 4], correlating well with the results of the protein modelling made in I-tasser.
  
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<img src=" https://2019.igem.org/wiki/images/thumb/d/d6/T--CU--tds1.png/800px-T--CU--tds1.png" alt"" width="500px" height="300px" ;" />
  
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<h2><b>Measuring cutA Binding Affinity</b></h2>
 
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We identified the best production time which is 14 hours, then we expressed the protein to test its affinity of binding to copper and sodium based on the modeling results, using TDS meter,we measured the decrease of 159g/l and 70g/l copper sulfate and sodium chloride,respectively. We made replicates and took the average, CutA was able to decrease NaCl by 2 parts per million. However, it has a much higher affinity to copper where it reduced the amount of Copper sulfate by 0.3 part per thousand.
 
  
  
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https://2019.igem.org/wiki/images/a/a3/T--CU--4.png
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Latest revision as of 19:41, 12 December 2019

CutA1

This part is the coding sequence for a trimerization domain - CutA1.

Usage and Biology

Characterisation by CU 2019 team

In silico protein modeling

First step in characterising CutA1(BBa_k245129), we used the Expasy ProtParam tool, where the theoretical protein extinction coefficient has been identified in addition to several other factors[Table1].

Extiction Coeficcient Gravy Molecular Weight PI Instability Index Aliphatic Index kDa
20690 -0.037 17791.29 5.40 40.03 "unstable" 85.84 12.321

Then we identified the protein 3D structure[fig.1] and the ligand it binds to[fig.2] using I-tasser. I-tasser was used to view the folding structure and predict its affinity to bind metal ligands in addition, to confirm the molecular function. where the model generated was closely related to Crystal structure of possible CutA1 divalent ion tolerance protein from Cryptosporidium parvum Iowa II, with TM-Score 0.95, and sequence coverage 0.9991.


Fig1.Predicted Model with the least free energy

Fig.2 Model showing protein binding to sodium ion with c-score: 0.1

The biological process identified in the model in response to metal ion, through the ligand binding site prediction,predicts that CutA1 could bind to sodium ion;Gene ontology predicted in the model belonged to copper binding.

CutA production using Linear expression cell-free kit

The protein produced using Ls70 linear expression cell-free kit, under the control of constitutive family member promotor (J23102 ) and strong RBS ( B0034). We identified protein concentration by measuring the absorbance at 280nm, concentration was calculated by beer’s law (A = εbc) using the theoretical extinction coefficient shown in Table1. We then plotted the time of reaction vs the concentration of the protein produced. It was found that the highest production was after 14hrs[fig.3]

Fig3.Showing CutA1 conc at different time intervals

Testing the binding affinities of CutA1.

We made a comparison between the ability of CutA1 protein to reduce both the TDS of Sodium salt and the Metal salt that it originally binds to; which is in this case Copper. So we measured the TDS before and after adding CutA1 to Copper Sulfate solution and Sodium Chloride Solution , We have found that CutA1 reduced the TDS of NaCl solution by approximately 900ppm [fig. 4], correlating well with the results of the protein modelling made in I-tasser.

Sequence and Features BBa_K245129 SequenceAndFeatures