Cloning of Acetoacetyl-CoA reductase and Polyhydroxybutyrate synthase genes from the local isolate Bacillus aryabhattai 6N-NRC into Escherichia coli
| Autor: | Abd El-Nasser A. Khattab, Magdy A. Amin, Reda F. Allam, Mona S Shafei, Yasser Ragab, Neveen M. El-Metwally |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Cloning ATP synthase biology Acetoacetyl-CoA reductase Chemistry 010501 environmental sciences medicine.disease_cause 01 natural sciences Polyhydroxybutyrate 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Biochemistry Acetoacetyl-CoA Gene expression medicine biology.protein Pharmacology (medical) Pharmacology Toxicology and Pharmaceutics (miscellaneous) Escherichia coli Gene 0105 earth and related environmental sciences |
| Zdroj: | Research Journal of Pharmacy and Technology. :3299-3306 |
| ISSN: | 0974-360X 0974-3618 |
| Popis: | Polyhydroxybutyrate (PHB) is the most known degradable biopolymer, produced by some genera of bacteria under unfavorable growth conditions. Isolation and cloning of acetoacetyl-CoA reductase (phbB) and polyhydroxybutyrate synthase (phbC) genes from local isolate previously identified as Bacillus aryabhattai 6N-NRC (GenBank accession no. MH997667.1) was achieved. Suitable primers designed for the phbB and phbC PCR approach were used to clone the phbB and phbC genes. The phbB and phbC genes were successfully isolated, cloned and the PCR amplicon 744 bp and 1089 bp corresponding to phbB and phbC genes were identified, cloned with the pET-29a (+) carrying the phbB and phbC genes, transformed and expressed in Escherichia coli BL21. The amplification of the phbB and phbC genes using specific primers of pET-29a (+) plasmid was performed. The open reading frame of phbB sequence was found to be 99.06% identical to the sequence of acetoacetyl-CoA reductase of B. aryabhattai (GenBank accession no. CP024035.1), while the open reading frame of phbC sequence was found to be 87.18% identical to the sequence of polyhydroxybutyrate synthase of B. aryabhattai (Gen Bank accession no. CP024035.1) after DNA sequencing. The analysis of the recombinant proteins from E. coli BL21 recombinant colony by tricine-polyacrylamide gel electrophoresis clarified that the expressed phbB and phbC genes in E. coli BL21 strain showed distinct bands of intensity 26.3 KD and 37.5 KD, respectively. |
| Databáze: | OpenAIRE |
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