The Escherichia coli motA Flagellar Gene as a Potential Integration Site for Large Synthetic DNA
| Autor: | Mario Juhas, Sheila Nathan, Orr Yarkoni, Kiew Lian Wan, Chee Hoo Yip, James W. Ajioka |
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| Rok vydání: | 2019 |
| Předmět: |
Genetics
Multidisciplinary Circular bacterial chromosome 05 social sciences Biology medicine.disease_cause law.invention Synthetic biology chemistry.chemical_compound Plasmid Chromosomal Insertion chemistry law 0502 economics and business medicine Recombinant DNA 050211 marketing Escherichia coli Gene 050203 business & management DNA |
| Zdroj: | Sains Malaysiana. 48:81-91 |
| ISSN: | 0126-6039 |
| DOI: | 10.17576/jsm-2019-4801-10 |
| Popis: | Escherichia coli is used as a chassis for many synthetic biology applications. However, the limitations of maintaining recombinant plasmids extra-chromosomally include increased metabolic burden to the host, constant selective pressure, variable plasmid copy number and plasmid instability that leads to curing. Hence, to overcome these limitations, DNA constructs are integrated into the bacterial chromosome to allow stable control of copy number and to reduce the metabolic burden towards the surrogate host. Non-essential E. coli flagellar genes have been proposed as potential chromosomal insertion target sites. In this study, we validated and compared the efficiency of two loci, namely motA and flgG, as target sites for synthetic biology applications. To enable this comparison, a dual reporter strain (DRS) that utilises two reporter proteins, EforRED and Venus, was developed as a test case. Initially, a yellow reporter plasmid k14.1_Venus was constructed and subsequently used as the plasmid backbone for the generation of two other plasmids, k14.1_eforRED and pcat_Venus, required to build the dual reporter strain. In the DRS, the eforRED gene was inserted into flgG whereas motA was disrupted by Venus. This mutant strain was defective in motility (p |
| Databáze: | OpenAIRE |
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