Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli
Genetic manipulations including chromosome engineering are essential techniques used to restructure cell metabolism. Lambda/Red (λ/Red)-mediated recombination is the most commonly applied approach for chromosomal modulation in Escherichia coli. However, the efficiency of this method is significantly...
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doaj-625e727dd021449b9e3a7bf942cddb5d2020-11-25T03:05:54ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852020-04-01810.3389/fbioe.2020.00145510415Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coliXinyi Xu0Xinyi Xu1Huichang Zhong2Weifeng Liu3Yong Tao4Engineering Research Center of Molecular and & Neuroimaging, Ministry of Education, School of Life Sciences and Technology, Xidian University, Xi’an, ChinaChinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, ChinaXiamen Huison Biotech Co., Ltd., Xiamen, ChinaChinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, ChinaChinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, ChinaGenetic manipulations including chromosome engineering are essential techniques used to restructure cell metabolism. Lambda/Red (λ/Red)-mediated recombination is the most commonly applied approach for chromosomal modulation in Escherichia coli. However, the efficiency of this method is significantly hampered by the laborious removal of the selectable markers. To overcome the problem, the integration helper plasmid was constructed, pSBC1a-CtR, which contains Red recombinase, Cre recombinase, and exogenous orthogonal aminoacyl-transfer RNA (tRNA) synthetase/tRNA pairs, allows an unnatural amino acid (UAA) to be genetically encoded at the defined site of the antibiotic resistance gene-encoded protein. When UAAs are not in the culture medium, there was no expression in the antibiotic resistance gene-encoded protein. Accordingly, the next procedure of antibiotic gene excising is not needed. To verify this method, poxB gene was knocked out successfully. Furthermore, sequential deletion of three target genes (galR, ptsG, and pgi) was able to generate neurosporene-producing strain marked by high growth rate. Thus, the site-specific incorporation UAA mutagenesis system were used to control and expand the use of conditional selectable marker, and the technique is used to facilitate a rapid continuous genome editing in Escherichia coli.https://www.frontiersin.org/article/10.3389/fbioe.2020.00145/fullunnatural amino acidsCre-mediated recombination systemantibiotic resistance geneneurosporeneEscherichia coli |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Xinyi Xu Xinyi Xu Huichang Zhong Weifeng Liu Yong Tao |
spellingShingle |
Xinyi Xu Xinyi Xu Huichang Zhong Weifeng Liu Yong Tao Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli Frontiers in Bioengineering and Biotechnology unnatural amino acids Cre-mediated recombination system antibiotic resistance gene neurosporene Escherichia coli |
author_facet |
Xinyi Xu Xinyi Xu Huichang Zhong Weifeng Liu Yong Tao |
author_sort |
Xinyi Xu |
title |
Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli |
title_short |
Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli |
title_full |
Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli |
title_fullStr |
Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli |
title_full_unstemmed |
Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli |
title_sort |
extension of genetic marker list using unnatural amino acid system: an efficient genomic modification strategy in escherichia coli |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Bioengineering and Biotechnology |
issn |
2296-4185 |
publishDate |
2020-04-01 |
description |
Genetic manipulations including chromosome engineering are essential techniques used to restructure cell metabolism. Lambda/Red (λ/Red)-mediated recombination is the most commonly applied approach for chromosomal modulation in Escherichia coli. However, the efficiency of this method is significantly hampered by the laborious removal of the selectable markers. To overcome the problem, the integration helper plasmid was constructed, pSBC1a-CtR, which contains Red recombinase, Cre recombinase, and exogenous orthogonal aminoacyl-transfer RNA (tRNA) synthetase/tRNA pairs, allows an unnatural amino acid (UAA) to be genetically encoded at the defined site of the antibiotic resistance gene-encoded protein. When UAAs are not in the culture medium, there was no expression in the antibiotic resistance gene-encoded protein. Accordingly, the next procedure of antibiotic gene excising is not needed. To verify this method, poxB gene was knocked out successfully. Furthermore, sequential deletion of three target genes (galR, ptsG, and pgi) was able to generate neurosporene-producing strain marked by high growth rate. Thus, the site-specific incorporation UAA mutagenesis system were used to control and expand the use of conditional selectable marker, and the technique is used to facilitate a rapid continuous genome editing in Escherichia coli. |
topic |
unnatural amino acids Cre-mediated recombination system antibiotic resistance gene neurosporene Escherichia coli |
url |
https://www.frontiersin.org/article/10.3389/fbioe.2020.00145/full |
work_keys_str_mv |
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