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Evaluation of Plasmid Transmission in Bacillus Subtilis by Cell Wall Modification

Mohamadzadeh, Mohammad Sadegh | 2021

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 54092 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Roosta Azad, Reza; Aghamollaei, Hossein
  7. Abstract:
  8. Today, almost all the country's needs in the field of industrial enzymes are met through imports. In this regard, much attention has been paid to the industrial production of various enzymes in the country. The production of recombinant proteins through strain engineering is one of the most important ways to meet this demand due to its many benefits. One of the most important microorganisms that is widely used in the enzyme industry is Bacillus subtilis. Due to its thick peptidoglycan wall, this gram-positive bacterium hardly accepts engineered carriers for genetic modification as hosts. The aim of this study was to evaluate the permeability of the wall and cell membrane of Bacillus subtilis to receive plasmid carriers. In this study, three different transformation methods were used to evaluate the transfer of plasmid to bacteria. In the first method, the combination of two methods of electroporation and the addition of CM11 antibacterial peptides to the bacterial medium was investigated. In this method, five different variables including 4 different culture media, 4 different recovery media, 4 different voltages in the electroporation device, 4 different recovery times and 2 different times to add polypeptides to the medium were evaluated. After obtaining the optimal value for each of the variables in the transformation process, the second method was performed using an electroporation device and the third method was using a polypeptide with the optimal values obtained from the first method. Finally, by increasing the permeability of the cell wall and cytoplasmic membrane of Bacillus subtilis, the highest efficiency of the transformation process was achieved in the most optimal cases of the first method in the simultaneous use of electroporation and antibacterial polypeptide at a rate of 1.69×104 CFU/µg DNA
  9. Keywords:
  10. Transformation ; Antibacterial Properties ; Electroporation ; Bacillus Subtilis ; Peptides ; Antibacterial Peptides ; Plasmid

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