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

Chee Hoo Yip, Orr Yarkoni, Mario Juhas, James Ajioka, Kiew Lian Wan, Sheila Nathan

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)81-91
Number of pages11
JournalSains Malaysiana
Volume48
Issue number1
DOIs
Publication statusPublished - 1 Jan 2019

Fingerprint

Venus
Plasmids
Synthetic Biology
Escherichia coli
DNA
Genes
Bacterial Chromosomes
Fluorescence
Growth
Proteins

Keywords

  • Chromosomal integration
  • Protein expression
  • Reporter system
  • Synthetic biology

ASJC Scopus subject areas

  • General

Cite this

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; Nathan, Sheila.

In: Sains Malaysiana, Vol. 48, No. 1, 01.01.2019, p. 81-91.

Research output: Contribution to journalArticle

Yip, Chee Hoo ; Yarkoni, Orr ; Juhas, Mario ; Ajioka, James ; Wan, Kiew Lian ; Nathan, Sheila. / The Escherichia coli mota flagellar gene as a potential integration site for large synthetic DNA. In: Sains Malaysiana. 2019 ; Vol. 48, No. 1. pp. 81-91.
@article{395575c09604415f935a088747287797,
title = "The Escherichia coli mota flagellar gene as a potential integration site for large synthetic DNA",
abstract = "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.",
keywords = "Chromosomal integration, Protein expression, Reporter system, Synthetic biology",
author = "Yip, {Chee Hoo} and Orr Yarkoni and Mario Juhas and James Ajioka and Wan, {Kiew Lian} and Sheila Nathan",
year = "2019",
month = "1",
day = "1",
doi = "10.17576/jsm-2019-4801-10",
language = "English",
volume = "48",
pages = "81--91",
journal = "Sains Malaysiana",
issn = "0126-6039",
publisher = "Penerbit Universiti Kebangsaan Malaysia",
number = "1",

}

TY - JOUR

T1 - The Escherichia coli mota flagellar gene as a potential integration site for large synthetic DNA

AU - Yip, Chee Hoo

AU - Yarkoni, Orr

AU - Juhas, Mario

AU - Ajioka, James

AU - Wan, Kiew Lian

AU - Nathan, Sheila

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - Chromosomal integration

KW - Protein expression

KW - Reporter system

KW - Synthetic biology

UR - http://www.scopus.com/inward/record.url?scp=85062269421&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85062269421&partnerID=8YFLogxK

U2 - 10.17576/jsm-2019-4801-10

DO - 10.17576/jsm-2019-4801-10

M3 - Article

VL - 48

SP - 81

EP - 91

JO - Sains Malaysiana

JF - Sains Malaysiana

SN - 0126-6039

IS - 1

ER -