Difference between revisions of "Part:BBa K3165048"
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Controller of Cell Division or Death B (CcdB) is the toxic component of the <i>Eschereschia coli</i> 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.<br>
 
Controller of Cell Division or Death B (CcdB) is the toxic component of the <i>Eschereschia coli</i> 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.<br>
−
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 ~ 42<sup>0</sup>C and that it aggregates at a higher temperature.<br>  
+
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 ~ 42<sup>o</sup>C and that it aggregates at a higher temperature.<br>  
 
We also used Top10 G (gryA) R462C which has a mutation in the Gyrase A at the 462<sup>th</sup> amino acid residue position i.e. change of arginine to cysteine which makes it resistant to recognition by CcdB (L83S).
 
We also used Top10 G (gryA) R462C which has a mutation in the Gyrase A at the 462<sup>th</sup> amino acid residue position i.e. change of arginine to cysteine which makes it resistant to recognition by CcdB (L83S).
  
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<p>
 
<p>
 
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 (500mL) was induced with 1g of 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 <b>leaky</b> transcription.
 
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 (500mL) was induced with 1g of 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 <b>leaky</b> transcription.
−
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>
+
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>
  
 
<<< IMAGE HERE >>> <br>
 
<<< IMAGE HERE >>> <br>
  
 
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.  
 
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.  
−
Quite evident from the gel, the size of the protein is around 20 KDa which is in range of the actual size of the monomeric fragment is 11.7 KDa.<br>  
+
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>  
  
 
<h3><b>Determination of the melting point of the Protein </b></h3>
 
<h3><b>Determination of the melting point of the Protein </b></h3>
−
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>0</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>0</sup>C. This can be confirmed from the graph.<br>
+
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>
  
 
<<< GRAPH HERE >>><br>
 
<<< GRAPH HERE >>><br>

Revision as of 16:15, 20 October 2019

ccdB (L83S) under araBAD

The CcdB toxin mutated to a highly unstable state so as to be expressed by common laboratory bacterial strains under arabinose activation.

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 Eschereschia 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 Eschereschia 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 GyraseA making the cells tolerant to it. The secondary culture (500mL) was induced with 1g of 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 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.
<<< IMAGE HERE >>>
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. 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.

Determination of the melting point of the Protein

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 63.8oC. We found, as expected, the CcdB L83S which has a core mutation making it less thermally stable and the melting point to 50oC. This can be confirmed from the graph.

<<< GRAPH HERE >>>

Size profiling of CcdB L83S by SEC (Size Exclusion Chromatography)

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.

<<< DATA >>>


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)"