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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/13056/1/10%20Chee-Hoo%20Yip.pdf
http://journalarticle.ukm.my/13056/
http://www.ukm.my/jsm/malay_journals/jilid48bil1_2019/KandunganJilid48Bil1_2019.html
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spelling my-ukm.journal.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
institution Universiti Kebangsaan Malaysia
building Perpustakaan Tun Sri Lanang Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Kebangsaan Malaysia
content_source UKM Journal Article Repository
url_provider http://journalarticle.ukm.my/
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/13056/1/10%20Chee-Hoo%20Yip.pdf
http://journalarticle.ukm.my/13056/
http://www.ukm.my/jsm/malay_journals/jilid48bil1_2019/KandunganJilid48Bil1_2019.html
_version_ 1643738966601498624
score 13.211869

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