The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA

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...

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Main Authors:	Yip, Chee-Hoo, Yarkoni, Orr, Juhas, Mario, Ajioka, James, Wan, Kiew-Lian, Sheila Nathan
Format:	Article
Language:	English
Published:	Penerbit Universiti Kebangsaan Malaysia 2019
Online Access:	http://journalarticle.ukm.my/13056/ http://journalarticle.ukm.my/13056/ http://journalarticle.ukm.my/13056/1/10%20Chee-Hoo%20Yip.pdf

id	ukm-13056
recordtype	eprints
spelling	ukm-130562019-06-20T15:15:09Z http://journalarticle.ukm.my/13056/ The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA Yip, Chee-Hoo Yarkoni, Orr Juhas, Mario Ajioka, James Wan, Kiew-Lian Sheila Nathan, 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<0.001) but growth rate was unaffected. The fluorescence emitted by Venus was higher (p<0.05) compared to EforRED, suggesting that motA is the better chromosomal target locus compared to flgG. Hence, this study proposes the use of E. coli motA as the site for chromosomal insertion for future synthetic biology applications. Penerbit Universiti Kebangsaan Malaysia 2019-01 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/13056/1/10%20Chee-Hoo%20Yip.pdf Yip, Chee-Hoo and Yarkoni, Orr and Juhas, Mario and Ajioka, James and Wan, Kiew-Lian and Sheila Nathan, (2019) The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA. Sains Malaysiana, 48 (1). pp. 81-91. ISSN 0126-6039 http://www.ukm.my/jsm/malay_journals/jilid48bil1_2019/KandunganJilid48Bil1_2019.html
repository_type	Digital Repository
institution_category	Local University
institution	Universiti Kebangasaan Malaysia
building	UKM Institutional Repository
collection	Online Access
language	English
description	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<0.001) but growth rate was unaffected. The fluorescence emitted by Venus was higher (p<0.05) compared to EforRED, suggesting that motA is the better chromosomal target locus compared to flgG. Hence, this study proposes the use of E. coli motA as the site for chromosomal insertion for future synthetic biology applications.
format	Article
author	Yip, Chee-Hoo Yarkoni, Orr Juhas, Mario Ajioka, James Wan, Kiew-Lian Sheila Nathan,
spellingShingle	Yip, Chee-Hoo Yarkoni, Orr Juhas, Mario Ajioka, James Wan, Kiew-Lian Sheila Nathan, The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA
author_facet	Yip, Chee-Hoo Yarkoni, Orr Juhas, Mario Ajioka, James Wan, Kiew-Lian Sheila Nathan,
author_sort	Yip, Chee-Hoo
title	The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA
title_short	The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA
title_full	The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA
title_fullStr	The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA
title_full_unstemmed	The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA
title_sort	escherichia coli mota flagellar gene as a potential integration site for large synthetic dna
publisher	Penerbit Universiti Kebangsaan Malaysia
publishDate	2019
url	http://journalarticle.ukm.my/13056/ http://journalarticle.ukm.my/13056/ http://journalarticle.ukm.my/13056/1/10%20Chee-Hoo%20Yip.pdf
first_indexed	2023-09-18T20:03:59Z
last_indexed	2023-09-18T20:03:59Z
_version_	1777407035975925760

The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA

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