Construction and characterization of a mutant outer membrane protein-A gene-deleted Escherichia coli strain_West China Medical Journal

Authors：

 YANG Xijing ¹ , CHENG Xiaoting ¹ , YU Simiao ² , LIU Wei ²

1. Animal Experiment Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. College of Life Sciences, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P. R. China;

Corresponding?author：

YANG Xijing, Email: yangxijingkelly@163.com

Keywords：

Outer membrane protein-A; Display system; Gene knock out

DOI：

10.7507/1002-0179.201701144

Video：

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Objective To construct an Escherichia coli outer membrane protein-A (OmpA) gene-deleted strain by Red homologous recombination, and laid the foundation for subsequent research. Methods Polymerase chain reaction (PCR) primers were designed according to the known OmpA gene sequence, and plasmid pKD3 for PCR amplification and integration; the fragment was transformed into Escherichia coli by λ-Red system in plasmid pKD46. After PCR checking and sequencing confirmation OmpA protein knocked out was observed by Western-blotting. Results The knock out gene product was correspond to a expected molecular weight. The western-blotting show that OmpA protein was knocked out. The difference in growth curve between the wild type and Escherichia coli △ OmpA gene-deleted strain was not significant. Conclusion OmpA gene deletion had no significant effect on the growth of Escherichia coli, which provides a foundation for further research on live vector vaccine.

Citation： YANG Xijing, CHENG Xiaoting, YU Simiao, LIU Wei. Construction and characterization of a mutant outer membrane protein-A gene-deleted Escherichia coli strain. West China Medical Journal, 2017, 32(12): 1895-1899. doi: 10.7507/1002-0179.201701144 Copy

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11.	楊汐靜, 張建新, 陳曉亭, 等. 應用 RED 同源重組技術構建表面展示鏈球菌 GapC1 的大腸埃希菌. 中國生物制品學雜志, 2015, 28(3): 239-244.
12.	Song B, Yang X, Sun H, et al. Immunogenicity of amino acids 1-150 of Streptococcus GapC displayed on the surface of Escherichia coli. Microb Pathog, 2017(105): 288-297.
13.	Lattemann CT, Maurer J, Gerland E, et al. Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter. J Bacteriol, 2000, 182(13): 3726-3733.
14.	Barillet F, Rupp R, Mignon-Grasteau S, et al. Genetic analysis for mastitis resistance and milk somatic cell score in French Lacaune dairy sheep. Genet Sel Evol, 2001, 33(4): 397-415.
15.	Wu ML, Tsai CY, Chen TH. Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity. FEMS Microbiol Lett, 2006, 256(1): 119-125.
16.	Majander K, Korhonen TK, Westerlund-Wikstrm B. Simultaneous display of multiple foreign peptides in the FliD capping and FliC filament proteins of the Escherichia coli flagellum. Appl Environ Microbiol, 2005, 71(8): 4263-4268.
17.	Yang C, Zhu Y, Yang J, et al. Development of an autofluorescent whole-cell biocatalyst by displaying dual functional moieties on Escherichia coli cell surfaces and construction of a coculture with organophosphate-mineralizing activity. Appl Environ Microbiol, 2008, 74(24): 7733-7739.
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1. 劉向昕, 展德文, 張兆山. 細菌表面展示技術的應用研究進展. 微生物學免疫學進展, 2005, 33(2): 70-74.
2. 孫軍德, 楊幼慧, 趙春燕. 微生物學. 南京: 東南大學出版社, 2009: 35-38.
3. Nhan NT, Gonzalez de Valdivia E, Gustavsson M, et al. Surface display of salmonella epitopes in Escherichia coli and Staphylococcus carnosus. Microb Cell Fact, 2011, 10: 22.
4. Lee SY, Choi JH, Xu Z. Microbial cell-surface display. Trends Biotechnol, 2003, 21(1): 45-52.
5. Karami A, Latifi AM, Khodi S. Comparison of the organophosphorus hydrolase surface display using InaVN and Lpp-OmpA systems in Escherichia coli. J Microbiol Biotechnol, 2014, 24(3): 379-385.
6. Earhart CF. Use of an Lpp-OmpA fusion vehicle for bacterial surface display. Methods Enzymol, 2000, 326(1): 506-516.
7. He MX, Feng H, Zhang YZ. Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif. Biotechnol Lett, 2008, 30(12): 2111-2117.
8. Hu S, Kong J, Sun Z, et al. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein. Microb Cell Fact, 2011, 10: 86.
9. Stathopoulos C, Georgiou G, Earhart CF. Characterization of escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane. Appl Microbiol Biotechnol, 1996, 45(1/2): 112-119.
10. 張建新, 湯煒, 周雪, 等. 大腸桿菌表面展示 Lpp’OmpA-TRAP 融合蛋白及其免疫原性. 第八屆全國會員代表大會暨第十五次學術研討會論文集. 徐州: 中國畜牧獸醫學會家畜傳染病學分會, 2013: 1214.
11. 楊汐靜, 張建新, 陳曉亭, 等. 應用 RED 同源重組技術構建表面展示鏈球菌 GapC1 的大腸埃希菌. 中國生物制品學雜志, 2015, 28(3): 239-244.
12. Song B, Yang X, Sun H, et al. Immunogenicity of amino acids 1-150 of Streptococcus GapC displayed on the surface of Escherichia coli. Microb Pathog, 2017(105): 288-297.
13. Lattemann CT, Maurer J, Gerland E, et al. Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter. J Bacteriol, 2000, 182(13): 3726-3733.
14. Barillet F, Rupp R, Mignon-Grasteau S, et al. Genetic analysis for mastitis resistance and milk somatic cell score in French Lacaune dairy sheep. Genet Sel Evol, 2001, 33(4): 397-415.
15. Wu ML, Tsai CY, Chen TH. Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity. FEMS Microbiol Lett, 2006, 256(1): 119-125.
16. Majander K, Korhonen TK, Westerlund-Wikstrm B. Simultaneous display of multiple foreign peptides in the FliD capping and FliC filament proteins of the Escherichia coli flagellum. Appl Environ Microbiol, 2005, 71(8): 4263-4268.
17. Yang C, Zhu Y, Yang J, et al. Development of an autofluorescent whole-cell biocatalyst by displaying dual functional moieties on Escherichia coli cell surfaces and construction of a coculture with organophosphate-mineralizing activity. Appl Environ Microbiol, 2008, 74(24): 7733-7739.
18. 楊汐靜. 展示在 XL1-Blue 大腸桿菌表面的鏈球菌 GapC_1-150aa 免疫原性研究. 大慶: 黑龍江八一農墾大學, 2014.

West China Medical Journal

Construction and characterization of a mutant outer membrane protein-A gene-deleted Escherichia coli strain

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