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Genome Engineering of Bacillus Subtilis Using CRISPR Technology to Overproduce Protease Production

Sabouri, Zahra | 2022

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 55138 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Roostaazad, Reza; Banaei Moghaddam, Ali Mohammad
  7. Abstract:
  8. Enzymes are an important part of detergents, they reduce the activation energy of the reaction and thus increase the efficiency of the process. Protease enzyme is one of the most common enzymes in detergents. Bacillus strain is the most important strain in the production of this enzyme and among the species of Bacillus, Bacillus subtilis is the most used. Bacillus subtilis is a gram-positive, rod-shaped bacterium. This bacterium is considered a non-pathogenic cell that has the ability to form spores and prevent death and damage in harsh conditions. In this study, CRISPR system was used to modify the genome of Bacillus subtilis ATCC 6633 in order to increase alkaline protease. To increase the production of this enzyme, using CRISPR, we replace the alkaline protease gene promoter with a stronger promoter.To increase the production of this enzyme, we use CRISPR to replace the alkaline protease gene promoter with a stronger promoter. This promoter was made and cloned into the CRISPR plasmid to modify the genome. The gRNA was also designed to guide cas9 endonuclease using bioinformatics tools and inserted into the CRISPR plasmid. Finally, the recombinant plasmid made for genome modification must be transferred to the bacterium Bacillus subtilis ATCC 6633
  9. Keywords:
  10. Alkaline Protease ; Bacillus Subtilis ; Promoter ; Genetic Engineering ; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)System ; Genome Editing

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