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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Abstract Full text Figures/Tables Video References Cited by

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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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.
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1. 刘向昕, 展德文, 张兆山. 细菌表面展示技术的应用研究进展. 微生物学免疫学进展, 2005, 33(2): 70-74.
2. 孙军德, 杨幼慧, 赵春燕. 微生物学. 南京: 东南大学出版社, 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. 张建新, 汤炜, 周雪, 等. 大肠杆菌表面展示 Lpp’OmpA-TRAP 融合蛋白及其免疫原性. 第八届全国会员代表大会暨第十五次学术研讨会论文集. 徐州: 中国畜牧兽医学会家畜传染病学分会, 2013: 1214.
11. 杨汐静, 张建新, 陈晓亭, 等. 应用 RED 同源重组技术构建表面展示链球菌 GapC1 的大肠埃希菌. 中国生物制品学杂志, 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. 杨汐静. 展示在 XL1-Blue 大肠杆菌表面的链球菌 GapC_1-150aa 免疫原性研究. 大庆: 黑龙江八一农垦大学, 2014.

West China Medical Journal

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

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