Difference between revisions of "Part:BBa K3165048"
 
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<partinfo>BBa_K3165048 short</partinfo>
 
<partinfo>BBa_K3165048 short</partinfo>
  
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The CcdB toxin mutated to a highly unstable state so as to be expressed by common laboratory bacterial strains under arabinose activation.
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The Controller of Cell Division or Death B (CcdB) toxin mutated to CcdB L83S <html><a href="https://parts.igem.org/Part:BBa_K3165014">(BBa_K3165014)</a></html> to reduce cytotoxicity under araBAD promoter <html><a  href="https://parts.igem.org/Part:BBa_K3165015">(BBa_K3165015)</a></html>.
  
 
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<p>
 
<p>
 
This device can be for the production CcdB L83S in Top10 G (gryA) R462C strain of <i>Escherichia coli
 
This device can be for the production CcdB L83S in Top10 G (gryA) R462C strain of <i>Escherichia coli
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</i>. Further, it can be used in Top10 PJAT strain with Gentamicin resistance for the production and to characterize it's effects on the bacterial population. The coding sequence of the CcdB L83S mutant protein is <html><a  href="https://parts.igem.org/Part:BBa_K3165014">(BBa_K3165014)</a></html> and under the promoter <html><a  href="https://parts.igem.org/Part:BBa_K3165015">(BBa_K3165015)</a></html>
+
</i>. Further, it can be used in Top10 PJAT strain with Gentamicin resistance for the production and to characterize it's effects on the bacterial population. The coding sequence of the CcdB L83S mutant protein is <html><a  href="https://parts.igem.org/Part:BBa_K3165014">(BBa_K3165014)</a></html> and under the promoter <html><a  href="https://parts.igem.org/Part:BBa_K3165015">(BBa_K3165015)</a></html>.
 
</p>
 
</p>
  
 
<h2> Characterisation </h2>
 
<h2> Characterisation </h2>
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<h3><b>IISc Bangalore 2019 </b></h3>
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<h3><b>IISc-Bangalore 2019 </b></h3>
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<h3><b>Expression and characterisation </b></h3>
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<h3><b>Expression and Characterisation </b></h3>
 
<p>
 
<p>
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This part was incorporated in Top10 G (gryA) R462C which is one of the strain that can be used to produce CcdB L83S because of the mutation in GyraseA making the cells tolerant to it. The secondary culture was induced with L-Arabinose and a total cell lysate was made by following the protein extraction protocol. Further, an uninduced sample was also separated as a control to check leaky transcription.
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This part was incorporated in Top10 G (gryA) R462C which is one of the strain that can be used to produce CcdB L83S because of the mutation in Gyrase A making the cells tolerant to it. <br>
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The supernatant is subjected to protein purification by incubation in the CcdA column and later eluted in 10 different samples which are loaded in the SDS PAGE mentioned. Further, for proper characterization, we have loaded the uninduced sample, total cell lysate, pellet, supernatant, flow-through, wash, ladder and 10 extracted elutes from right to left. <br>
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The secondary culture was induced with L-Arabinose and a total cell lysate was made by following the protein extraction protocol. Further, an uninduced sample was also separated as a control to check leaky transcription.
 +
The supernatant is subjected to protein purification by incubation in the CcdA column and later eluted to get 10 different samples which are loaded in the SDS PAGE mentioned. <br>
 
<html>
 
<html>
 
<figure>
 
<figure>
 
<center><div class="thumbnail">
 
<center><div class="thumbnail">
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                         <img src="https://static.igem.org/mediawiki/parts/4/4b/T--IISc-Bangalore--ccdB_gel1.png" style="width:70%;">
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                         <img src="https://static.igem.org/mediawiki/parts/4/4b/T--IISc-Bangalore--ccdB_gel1.png" style="width:90%;">
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             </div> <figcaption>Fig.1 - Trulli, Puglia, Italy.</figcaption> </center>
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             </div> <figcaption><i><b>Fig.1</b> - SDS PAGE Gel 1 showing the elutes for the purified <b>CcdB L83S</b> mutant protein.</i><br> </figcaption> </center>
 
  <br>
 
  <br>
 
</figure>
 
</figure>
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<figure>
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<center><div class="thumbnail">
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                        <img src="https://static.igem.org/mediawiki/parts/b/b5/T--IISc-Bangalore--ccdB_gel2.jpg" style="width:90%;">
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            </div> <figcaption><i><b>Fig.2</b> - SDS PAGE Gel 2 showing the elutes for the purified <b>CcdB L83S</b> mutant protein.</i><br> </figcaption> </center>
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<br>
 +
</figure>
 +
 +
 +
It can be seen from the SDS PAGE image that there is a very faint band in the well containing the uninduced sample. So, we concluded a very low level of leaky transcription which further can be reduced by the addition of glucose to the culture as glucose acts as a repressor. <br>
 +
Also, the faint band in the pellet section can be used to evaluate the quality of the protein extraction.
 +
As quite evident from the gel, the size of the protein is 11.7 kDa which matches the theoretical value.<br>
 +
 +
<h3><b>Determination of the melting point of the CcdB L83S Protein </b></h3>
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We also determined the melting point of the CcdB L83S mutant by performing Thermal Shift Assay.<br>
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<div class="container-fluid">
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  <div class="row">
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    <div class="col-sm-6">
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<figure>
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<center><div class="thumbnail">
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                        <img src="https://static.igem.org/mediawiki/parts/8/89/T--IISc-Bangalore--ccdB_melting_total_perfect.png" style="width:95%;">
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            </div> <figcaption><i><b>Fig.3</b> The full Thermal Shift Assay graph for determining the melting point of <b>CcdB L83S</b> mutant protein.</i><br> </figcaption> </center>
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<br>
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</figure>
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</div>
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    <div class="col-sm-6">
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<figure>
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<center><div class="thumbnail">
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                        <img src="https://static.igem.org/mediawiki/parts/1/15/T--IISc-Bangalore--ccdB_mean_partial_fluore.png" style="width:95%;">
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            </div> <figcaption><i><b>Fig.4</b> The zoomed in picture of the plot as mention in "Fig.3" Thermal Assay graph for determining the melting point of <b>CcdB L83S</b> mutant protein.</i><br> </figcaption> </center>
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<br>
 +
</figure>
 +
 +
</div>
 +
  </div>
 +
</div>
 
</html>
 
</html>
  
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It can be seen from the SDS PAGE image that there is a very faint band in the well containing the uninduced sample. So, we concluded that there is a very low level of leaky transcription happening which further can be reduced by the addition of glucose to the culture as glucose acts as a repressor.  
+
As can be seen from Fig.3 and Fig.4, the melting point of the <b>CcdB L83S</b> mutant protein is calculated to be 43.03<sup>o</sup>C. When compared to the melting point of the wild type CcdB protein i.e. 63.8<sup>o</sup>C, the CcdB L83S protein has a much lower melting temperature. Hence, we concluded that the core mutation resulted in the instability of the CcdB L83S mutant.<br>
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Quite evident from the gel, the size of the protein is around 12 KDa which is in range of the actual size of the monomeric fragment is 11.7 KDa.<br>
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In addition to this, Growth Assay can be used on strains like Top10 PJAT to draw the same conclusion i.e. the mutant CcdB L83S is less stable than it's wild type. So, this mutant can be used in regulating the bacterial population to an extent.
  
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<h3><b>Determination of the melting point of the Protein </b></h3>
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<h3><b>Growth Assay of CcdB L83S Protein mutant</b></h3>
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We also characterized the melting point of the CcdB L83S mutant by performing Protein Thermal Shift Assay. While the melting point of wild type CcdB is around 63.8<sup>o</sup>C. We found, as expected, the CcdB L83S which has a core mutation making it less thermally stable and the melting point to 50<sup>o</sup>C. This can be confirmed from the graph.<br>
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<<< GRAPH HERE >>><br>
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To show that CcdB L83S is not as potent as the wild type protein, Top 10 pJET strain of <i>E. coli</i> containing the CcdB L83S gene was grown on LB agar plates with gentamicin and ampicillin resistance and containing various concentrations of arabinose and glucose.<br>
  
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<h3><b>Size profiling of CcdB L83S by SEC (Size Exclusion Chromatography)</b></h3>
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<html>
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The exact size of the protein (Tertiary Structure) has been characterized by the data obtained from protein profiling done by Size Exclusion Chromatography. The data clearly states the protein to be a Dimer with a size of 23.4 kDa. <br>
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<figure>
 +
<center><div class="thumbnail">
 +
                        <img src="https://static.igem.org/mediawiki/parts/1/19/T--IISc-Bangalore--zero.jpg" style="width:35%;"><br><br>
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            </div> <figcaption><i><b>Fig.5</b> - The plate shows the colonies of pJAT strain grown with no amount of arabinose or glucose.</i><br> </figcaption> </center>
 +
<br>
 +
</figure>
 +
 
 +
We can see in <b>Fig.5</b> that when LB agar plate with no inducer or repressor was used, colonies of bacteria were obtained, which validated the hypothesis that the CcdB L83S mutant can be grown in strains of <i>E. coli</i> which have no GyraseA subunit mutation. <br>
 +
 
 +
<figure>
 +
<center><div class="thumbnail">
 +
                        <img src="https://static.igem.org/mediawiki/parts/8/8e/T--IISc-Bangalore--induction_with_glucose.png" style="width:69%;"><br><br>
 +
            </div> <figcaption><i><b>Fig.6</b> - The plates shown have a gradually decreasing concentration of glucose towards the left. A trend can be seen that the colony size increases from left to right.</i><br> </figcaption> </center>
 +
<br>
 +
</figure>
 +
 
 +
As can be seen from <b>Fig.6</b>, in the presence of glucose, the bacteria grew with at its normal rate and no effects were seen. It can further be noticed that the spots were more prominent at higher concentrations of glucose, which is valid as glucose is a repressor and it reduces the leaky transcription of the CcdB L83S protein.<br>
 +
 
 +
 
 +
<figure>
 +
<center><div class="thumbnail">
 +
                        <img src="https://static.igem.org/mediawiki/parts/8/8a/T--IISc-Bangalore--arabinose_plates.png" style="width:69%;"><br><br>
 +
            </div> <figcaption><i><b>Fig.7</b> - The plates shown have a gradually increasing concentration of arabinose towards the right. A trend can be seen that the colony size idecreases from left to right.</i><br> </figcaption> </center>
 +
<br>
 +
</figure>
  
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<<< DATA >>> <br>
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From <b>Fig.7</b>, we can see that despite the presence of arabinose in plates, we were able to visualize very small-sized colonies. But, at higher concentrations of arabinose, these colonies weren't able to grow. <br>
 +
This shows that CcdB L83S can function as a bacteriostatic protein or a bactericidal toxin under the right conditions, and can be used in strains of <i>E. coli</i> not containing mutations in Gyrase A subunit.
  
  
Line 63: Line 126:
 
"Protein Model Discrimination Using Mutational Sensitivity Derived from Deep Sequencing" <br>
 
"Protein Model Discrimination Using Mutational Sensitivity Derived from Deep Sequencing" <br>
 
https://doi.org/10.1016/j.str.2011.11.021<br>
 
https://doi.org/10.1016/j.str.2011.11.021<br>
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<li>Kanika BAJAJ, Ghadiyaram CHAKSHUSMATHI, Kiran BACHHAWAT-SIKDER, Avadhesha SUROLIA and Raghavan VARADARAJAN <br>
+
<li>Kanika Bajaj, Ghadiyaram Chakshusmathi, Kiran Bachhawat-Sikder, Avadhesha Surolia and Raghavan Varadarajan <br>
 
"Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)"
 
"Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)"
 
</li>
 
</li>

Latest revision as of 01:38, 22 October 2019

ccdB (L83S) under araBAD

The Controller of Cell Division or Death B (CcdB) toxin mutated to CcdB L83S (BBa_K3165014) to reduce cytotoxicity under araBAD promoter (BBa_K3165015).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 120
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 396


Usage and Biology

Biology

Controller of Cell Division or Death B (CcdB) is the toxic component of the Escherichia coli CcdAB anti-toxin toxin system. It is a globular, dimeric protein with 101 residues per protomer, involved in the maintenance of F plasmid in cells by a mechanism involving its binding to and the poisoning of DNA Gyrase which leads to the breaking of the double-stranded DNA in the bacteria. The L83S mutant of the wild type CcdB protein is being mutated at the 83rd amino acid residue position from Leucine to Serine in the core region of the protein.
Due to the core mutation in the wild type CcdB, the L83S mutant is highly unstable. This can be further verified by the data received from the Thermal Assay which confers it's melting point to be around ~ 42oC and that it aggregates at a higher temperature.
We also used Top10 G (gryA) R462C which has a mutation in the Gyrase A at the 462th amino acid residue position i.e. change of arginine to cysteine which makes it resistant to recognition by CcdB (L83S).

Usage

This device can be for the production CcdB L83S in Top10 G (gryA) R462C strain of Escherichia coli . Further, it can be used in Top10 PJAT strain with Gentamicin resistance for the production and to characterize it's effects on the bacterial population. The coding sequence of the CcdB L83S mutant protein is (BBa_K3165014) and under the promoter (BBa_K3165015).

Characterisation

IISc-Bangalore 2019

Expression and Characterisation

This part was incorporated in Top10 G (gryA) R462C which is one of the strain that can be used to produce CcdB L83S because of the mutation in Gyrase A making the cells tolerant to it.
The secondary culture was induced with L-Arabinose and a total cell lysate was made by following the protein extraction protocol. Further, an uninduced sample was also separated as a control to check leaky transcription. The supernatant is subjected to protein purification by incubation in the CcdA column and later eluted to get 10 different samples which are loaded in the SDS PAGE mentioned.

Fig.1 - SDS PAGE Gel 1 showing the elutes for the purified CcdB L83S mutant protein.

Fig.2 - SDS PAGE Gel 2 showing the elutes for the purified CcdB L83S mutant protein.

It can be seen from the SDS PAGE image that there is a very faint band in the well containing the uninduced sample. So, we concluded a very low level of leaky transcription which further can be reduced by the addition of glucose to the culture as glucose acts as a repressor.
Also, the faint band in the pellet section can be used to evaluate the quality of the protein extraction. As quite evident from the gel, the size of the protein is 11.7 kDa which matches the theoretical value.

Determination of the melting point of the CcdB L83S Protein

We also determined the melting point of the CcdB L83S mutant by performing Thermal Shift Assay.
Fig.3 The full Thermal Shift Assay graph for determining the melting point of CcdB L83S mutant protein.

Fig.4 The zoomed in picture of the plot as mention in "Fig.3" Thermal Assay graph for determining the melting point of CcdB L83S mutant protein.

As can be seen from Fig.3 and Fig.4, the melting point of the CcdB L83S mutant protein is calculated to be 43.03oC. When compared to the melting point of the wild type CcdB protein i.e. 63.8oC, the CcdB L83S protein has a much lower melting temperature. Hence, we concluded that the core mutation resulted in the instability of the CcdB L83S mutant.
In addition to this, Growth Assay can be used on strains like Top10 PJAT to draw the same conclusion i.e. the mutant CcdB L83S is less stable than it's wild type. So, this mutant can be used in regulating the bacterial population to an extent.

Growth Assay of CcdB L83S Protein mutant

To show that CcdB L83S is not as potent as the wild type protein, Top 10 pJET strain of E. coli containing the CcdB L83S gene was grown on LB agar plates with gentamicin and ampicillin resistance and containing various concentrations of arabinose and glucose.



Fig.5 - The plate shows the colonies of pJAT strain grown with no amount of arabinose or glucose.

We can see in Fig.5 that when LB agar plate with no inducer or repressor was used, colonies of bacteria were obtained, which validated the hypothesis that the CcdB L83S mutant can be grown in strains of E. coli which have no GyraseA subunit mutation.


Fig.6 - The plates shown have a gradually decreasing concentration of glucose towards the left. A trend can be seen that the colony size increases from left to right.

As can be seen from Fig.6, in the presence of glucose, the bacteria grew with at its normal rate and no effects were seen. It can further be noticed that the spots were more prominent at higher concentrations of glucose, which is valid as glucose is a repressor and it reduces the leaky transcription of the CcdB L83S protein.


Fig.7 - The plates shown have a gradually increasing concentration of arabinose towards the right. A trend can be seen that the colony size idecreases from left to right.

From Fig.7, we can see that despite the presence of arabinose in plates, we were able to visualize very small-sized colonies. But, at higher concentrations of arabinose, these colonies weren't able to grow.
This shows that CcdB L83S can function as a bacteriostatic protein or a bactericidal toxin under the right conditions, and can be used in strains of E. coli not containing mutations in Gyrase A subunit.

References

  1. Anusmita Sahoo, Shruti Khare, Sivasankar Devanarayanan, Pankaj C. Jain, and Raghavan Varadarajan
    "Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis"
    doi: 10.7554/eLife.09532
  2. Bharat V.Adkar, Arti Tripathi, Anusmita Sahoo, Kanika Bajaj, Devrishi Goswami, Purbani Chakrabarti, Mohit K. Swarnkar, Rajesh S.Gokhale, Raghavan Varadarajan
    "Protein Model Discrimination Using Mutational Sensitivity Derived from Deep Sequencing"
    https://doi.org/10.1016/j.str.2011.11.021
  3. Kanika Bajaj, Ghadiyaram Chakshusmathi, Kiran Bachhawat-Sikder, Avadhesha Surolia and Raghavan Varadarajan
    "Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)"