Sains Malaysiana 48(1)(2019): 81–91

http://dx.doi.org/10.17576/jsm-2019-4801-10

The Escherichia coli motA Flagellar Gene as a Potential Integration Site for Large Synthetic DNA

(Gen Flagelum Escherichia coli motA sebagai Tapak Integrasi yang Berpotensi untuk DNA Sintetik Besar)

CHEE-HOO YIP^1,2, ORR YARKONI², MARIO JUHAS², JAMES AJIOKA², KIEW-LIAN WAN¹ & SHEILA NATHAN¹*

¹School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Department of Pathology, Tennis Court Road, University of Cambridge, CB2 1QP Cambridge, United Kingdom

Received: 29 March 2018/Accepted: 28 August 2018

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

ABSTRAK

Bakteria Escherichia coli digunakan sebagai kes dalam banyak aplikasi biologi sintetik. Walau bagaimanapun, cabaran untuk mengekalkan plasmid rekombinan di luar kromosom termasuk peningkatan beban metabolik kepada perumah, tekanan memilih yang berterusan, pelbagai bilangan salinan plasmid dan ketidakstabilan plasmid membawa kepada penyingkiran plasmid daripada bakteria. Untuk mengatasi batasan tersebut, binaan DNA diintegrasikan ke dalam kromosom bakteria untuk membenarkan bilangan salinan gen yang terkawal dan mengurangkan beban metabolik kepada perumah pengganti. Gen flagelum yang tidak perlu telah dicadangkan sebagai tapak sasaran penyisipan kromosom yang berpotensi. Dalam kajian ini, kami mengesah dan membandingkan kecekapan dua lokus, iaitu motA dan flgG, sebagai tapak sasaran untuk aplikasi biologi sintetik. Untuk membenarkan perbandingan ini, strain dwipelapor (DRS) yang menggunakan dua protein pelapor, EforRED dan Venus, telah dibangunkan sebagai kes ujian. Pada mulanya, plasmid pelapor kuning, k14.1_Venus dibina dan kemudiannya digunakan sebagai tulang belakang plasmid untuk menjana dua plasmid lain, k14.1_eforRED dan pcat_Venus, yang diperlukan untuk membina DRS. Dalam DRS, gen eforRED diselitkan ke dalam flgG manakala motA disisip dengan Venus. Kemortilan strain mutan ini dimansuhkan (p<0.001) tetapi kadar pertumbuhannya tidak terjejas. Pendarfluor yang dipancarkan oleh Venus lebih tinggi (p<0.05) berbanding dengan EforRED, menunjukkan bahawa motA merupakan lokus sasaran kromosom yang lebih baik berbanding dengan flgG. Oleh itu, kajian ini mencadangkan penggunaan E. coli motA sebagai tapak untuk penyisipan kromosom dalam aplikasi biologi sintetik pada masa depan.

Kata kunci: Biologi sintetik; integrasi kromosom; pengungkapan protein; sistem pelapor

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*Corresponding author; email: sheila@ukm.edu.my