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 YIP1,2, 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

 

Diserahkan: 29 Mac 2018/Diterima: 28 Ogos 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

RUJUKAN

Ajikumar, P.K., Xiao, W., Tyo, K.E.J., Wang, Y., Simeon, F., Leonard, E., Mucha, O., Phon, T.H., Pfeifer, B. & Stephanopoulos, G. 2010. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330(6000): 70-74.

Alieva, N.O., Konzen, K.A., Field, S.F., Meleshkevitch, E.A., Hunt, M.E., Salih, A. & Matz, M.V. 2008. Diversity and evolution of coral fluorescent proteins. PLoS ONE 3(7): 1-12.

Atsumi, S., Hanai, T. & Liao, J.C. 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451: 86-89.

Bai Flagfeldt, D., Siewers, V., Huang, L. & Nielsen, J. 2009. Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae. Yeast 26(10): 545-551.

Bentley, W.E. & Kompala, D.S. 1990. Optimal induction of protein synthesis in recombinant bacterial cultures. Annals of the New York Academy of Sciences 589(1): 121-138.

Bhattacharya, S.K. & Dubey, A.K. 1995. Metabolic burden as reflected by maintenance coefficient of recombinant Escherichia coli overexpressing target gene. Biotechnology Letters 17(11): 1155-1160.

Bloemendal, S., Löper, D., Terfehr, D., Kopke, K., Kluge, J., Teichert, I. & Kück, U. 2014. Tools for advanced and targeted genetic manipulation of the β-lactam antibiotic producer Acremonium chrysogenum. Journal of Biotechnology 169: 51-62.

Carneiro, S., Ferreira, E.C. & Rocha, I. 2013. Metabolic responses to recombinant bioprocesses in Escherichia coli. Journal of Biotechnology 164(3): 396-408.

Chang, M.C.Y., Eachus, R.A., Trieu, W., Ro, D.K. & Keasling, J.D. 2007. Engineering Escherichia coli for the production of functionalized terpenoids using plant P450s. Nature Chemical Biology 3: 274-277.

Cho, K.M., Yoo, Y.J. & Kang, H.S. 1999. δ-Integration of endo/exo-glucanase and β-glucosidase genes into the yeast chromosomes for direct conversion of cellulose to ethanol. Enzyme and Microbial Technology 25(1-2): 23-30.

Cunningham, D.S., Koepsel, R.R., Ataai, M.M. & Domach, M.M. 2009. Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint. Microbial Cell Factories 17: 1-17.

Datsenko, K.A. & Wanner, B.L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences USA 97(12): 6640-6645.

Davis, M.W. ApE: A plasmid editor. 2012. Version 2.0.49. Utah: University of Utah.

Gibson, D.G., Young, L., Chuang, R., Venter, J.C., Hutchison, C. & Smith, H.O. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6(5): 343-345.

Glick, B.R. 1995. Metabolic load and heterologous gene expression. Biotechnology Advances 13(2): 247-261.

Gu, P., Yang, F., Su, T., Wang, Q., Liang, Q. & Qi, Q. 2015. A rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies. Scientific Reports 5(9684): 1-9.

Hanahan, D., Jessee, J. & Bloom, F.R. 1991. Plasmid transformation of Escherichia coli and other bacteria. Methods in Enzymology 204: 63-113.

Haseloff, J. & Ajioka, J. 2009. Synthetic biology: History, challenges and prospects. Journal of the Royal Society, Interface/the Royal Society 6(6): 389-391.

Ishikawa, M. & Hori, K. 2013. A new simple method for introducing an unmarked mutation into a large gene of non-competent gram-negative bacteria by FLP/FRT recombination. BMC Microbiology 13(86): 1-10.

Juhas, M. 2016. On the road to synthetic life: The minimal cell and genome-scale engineering. Critical Reviews in Biotechnology 36(3): 416-423.

Juhas, M. & Ajioka, J.W. 2015a. Identification and validation of novel chromosomal integration and expression loci in Escherichia coli flagellar region 1. PLoS ONE 10(3): 1-13.

Juhas, M. & Ajioka, J.W. 2015b. Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions. Microbial Biotechnology 8(4): 726-738.

Juhas, M., Evans, L.D.B., Frost, J., Davenport, P.W., Yarkoni, O., Fraser, G.M. & Ajioka, J.W. 2014. Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits. PLoS ONE 9(10): 1-7.

Juhas, M., Davenport, P.W., Brown, J.R., Yarkoni, O. & Ajioka, J.W. 2013. Meeting report: The Cambridge BioDesign TechEvent - Synthetic Biology, a new 'Age of Wonder'? BTJ-FORUM. pp. 761-763.

Keasling, J.D. 2008. Synthetic biology for synthetic chemistry. ACS Chemical Biology 3: 64-76.

Kuhlman, T.E. & Cox, E.C. 2010. Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Research 38(6): 1-10.

Kulkarni, S.K. & Stahl, F.W. 1989. Interaction between the sbcC gene of Escherichia coli and the gam gene of phage λ. Genetics 253: 249-253.

Macnab, R.B. 2003. How bacteria assemble flagellar. Annual Review of Microbiology 57(1): 77-100.

Martinez-Morales, F., Borges, A.C., Martinez, A., Shanmugam, K.T. & Ingram, L.O. 1999. Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction. Journal of Bacteriology 181(22): 7143-7148.

Mosberg, J.A., Lajoie, M.J. & Church, G.M. 2010. Lambda Red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186(3): 791-799.

Muniyappa, K. & Radding, C.M. 1986. The homologous recombination system of phage λ: Pairing activities of β protein. Journal of Biological Chemistry 261: 7472-7478.

Murphy, K.C. & Campellone, K.G. 2003. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Molecular Biology 4: 11.

Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K. & Miyawaki, A. 2002. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnology 20(1): 87-90.

Ringrose, L., Lounnas, V., Ehrlich, L., Buchholz, F., Wade, R. & Stewart, A.F. 1998. Comparative kinetic analysis of FLP and cre recombinases: Mathematical models for DNA binding and recombination. Journal of Molecular Biology 284(2): 363-384.

Silva, F., Queiroz, J.A. & Domingues, F.C. 2012. Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli. Biotechnology Advances 30(3): 691-708.

Sabri, S., Steen, J.A., Bongers, M., Nielsen, L.K. & Vickers, C.E. 2013. Knock-in/knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci. Microbial Cell Factories 12(60): 1-14.

Takekawa, N., Terahara, N., Kato, T. & Gohara, M. 2016. The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium. Scientific Reports 6(31526): 1-8.

Tyo, K.E., Ajikumar, P.K. & Stephanopoulos, G. 2009. Stabilized gene duplication enables long-term selection-free heterologous pathway expression. Nature Biotechnology 27(8): 760-765.

Ublinskaya, A.A., Samsonov, V.V., Mashko, S.V. & Stoynova, N.V. 2012. A PCR-free cloning method for the targeted Q80 Int-mediated integration of any long DNA fragment, bracketed with meganuclease recognition sites, into the Escherichia coli chromosome. Journal of Microbiological Methods 89: 167-173.

van der Krogt, G.N., Ogink, J., Ponsioen, B. & Jalink, K. 2008. A comparison of donor-acceptor pairs for genetically encoded FRET sensors: Application to the Epac cAMP sensor as an example. PLoS ONE 3(4): 1-9.

Yansura, D.G. & Henner, D.J. 1984. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proceedings of the National Academy of Sciences USA 81(2): 439-443.

Zhu, X.D. & Sadowski, P.D. 1995. Cleavage-dependent ligation by the FLP recombinase. Journal of Biological Chemistry 270(39): 23044-23054.

 

*Pengarang untuk surat-menyurat; email: sheila@ukm.edu.my  

 

 

 

 

 

sebelumnya