Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli

• Published in: Frontiers in bioengineering and biotechnology 2020-04, Vol.8, p.145-145
• Main Authors: Xu, Xinyi, Zhong, Huichang, Liu, Weifeng, Tao, Yong
• Format: Article
• Language: English
• Subjects: antibiotic resistance gene; Bioengineering and Biotechnology; Cre-mediated recombination system; Escherichia coli; neurosporene; unnatural amino acids
• ISSN: 2296-4185; 2296-4185
• DOI: 10.3389/fbioe.2020.00145

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 . 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, B gene was knocked out successfully. Furthermore, sequential deletion of three target genes ( R, G, and ) 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 .