利用响应面法优化发酵乳杆菌AR497电转化条件
|
摘要
以1株能有效改善小鼠肠炎的发酵乳杆菌AR497为研究对象,在Plackett-Burman试验的基础上选取甘氨酸质量浓度、OD_(600)、电场强度和山梨醇浓度为自变量,电转化效率为响应值,利用Box-Behnken响应面法优化其电转化条件。最优条件为:电场强度15 kV/cm,山梨醇浓度0.6 mol/L,OD_(600)0.2,甘氨酸质量浓度26 g/L。在此条件下,电转化效率高达2.78×10~5 CFU/μg DNA,比优化前提高了22倍,实现了发酵乳杆菌AR497的高效遗传转化,为后续对其进行遗传改造和解析抗炎机制奠定基础。
Lactobacillus fermentum AR497 that can evidently relieve intestinal inflammation in mice was used as the main subject. Based on the Plackett-Burman tests, glycine concentrations, OD_(600), electric field strength, and sorbitol concentrations were selected as independent variables, the electroporation efficiency was the response value. Further optimization was made by using Box-Behnken response surface methodology. The optimum values for electric field strength,sorbitol content,OD_(600), and glycine content were 15 kV/cm,0.6 mol/L,0.2 and 26 g/L, respectively. The transformants reached 2.78×10~5 CFU/μg DNA under the above conditions, which was improved about 22-fold compared with that of unoptimized condition. The highly frequent genetic transformation of AR497 was realized in this work, which laid the foundation for its genetic editing and elucidating its anti-inflammation mechanism.
Lactobacillus fermentum AR497 that can evidently relieve intestinal inflammation in mice was used as the main subject. Based on the Plackett-Burman tests, glycine concentrations, OD_(600), electric field strength, and sorbitol concentrations were selected as independent variables, the electroporation efficiency was the response value. Further optimization was made by using Box-Behnken response surface methodology. The optimum values for electric field strength,sorbitol content,OD_(600), and glycine content were 15 kV/cm,0.6 mol/L,0.2 and 26 g/L, respectively. The transformants reached 2.78×10~5 CFU/μg DNA under the above conditions, which was improved about 22-fold compared with that of unoptimized condition. The highly frequent genetic transformation of AR497 was realized in this work, which laid the foundation for its genetic editing and elucidating its anti-inflammation mechanism.
引文
[1] MIKELSAAR M ,ZILMER M .Lactobacillus fermentum ME-3-an antimicrobial and antioxidative probiotic[J].Microbial Ecology in Health and Disease,2009,21(1):1-27.
[2] SUN Z,LIU W,GAO W,et al.Identification and characterization of the dominant lactic acid bacteria from kurut:the naturally fermented yak milk in Qinghai,China[J].The Journal of General and Applied Microbiology,2010,56(1):1-10.
[3] LIU S N,HAN Y,ZHOU Z J.Lactic acid bacteria in traditional fermented Chinese foods[J].Food Research International,2011,44(3):643-651.
[4] LJUNGH A,WADSTR?M T.Lactic acid bacteria as probiotics[J].Current Issues In Microbiology,2006,7(2):73-89.
[5] KLAENHAMMER T R,ALTERMANN E,PFEILER E,et al.Functional genomics of probiotic Lactobacilli[J].Journal of Clinical Gastroenterology,2008,42(8):S160.
[6] 敖晓琳,蒲彪,蔡义民,等.发酵乳杆菌及其益生特性研究进展[J].食品与生物技术学报,2015(2):121-127.
[7] 王巧惠,王光强,宋馨,等.不同质粒电转三种乳酸菌的比较研究[J].工业微生物,2016,46(1):36-41.
[8] 凌代文,东秀珠.乳酸细菌分类鉴定及实验方法[J].微生物学通报,1998(1):23.
[9] EWE J A,WAN-ABDULLAH W N,ALIAS A K,et al.Bioconversion of isoflavones and the probiotic properties of the electroporated parent and subsequent three subcultures of Lactobacillus fermentum BT8219 in biotin-soymilk[J].Journal of Microbiology and Biotechnology,2012,22(22):947-959.
[10] RUSH C M,HAFNER L M,TIMMS P.Genetic modification of a vaginal strain of Lactobacillus fermentum and its maintenance within the reproductive tract after intravaginal administration[J].Journal of Medical Microbiology,1994,41(4):272.
[11] 郭兴华,曹郁生,东秀珠.益生乳酸细菌—分子生物学及生物技术[M].北京:科学出版社,2008.
[12] IRAM M,WANG X,ZHAO H Y,et al.Ameliorate maneuver for transformation of Lactobacillus strains by electroporation with IBDV-vp2 chemically engineered expression vector[J].Journal of Animal and Plant Sciences,2016,26(3):814-822.
[13] LUCHANSKY J B,MURIANA P M,KLAENHAMMER T R.Application of electroporation for transfer of plasmid DNA to Lactobacillus,Lactococcus,Leuconostoc,Listeria,Pediococcus,Bacillus,Staphylococcus,Enterococcus and Propionibacterium[J].Molecular Microbiology,1988,2(5):637-646.
[14] KIM Y H,HAN K S,OH S,et al.Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation[J].Journal of Applied Microbiology,2010,99(1):167-174.
[15] 张旭,崔艳华,张兰威,等.乳酸菌电转化条件研究[J].兰州大学学报(自科版),2009,45(s1):26-33.
[16] 曹晓梅,张虎成,李曼,等.乳杆菌电转化的研究进展[J].军事医学,2008,32(6):590-593.
[17] 万谦.乳酸菌质粒提取方法及电转化条件的研究[D].郑州:河南工业大学,2010.
[18] 夏子芳,石贵阳,张梁,等.乳杆菌电转化条件的优化[J].食品科学,2008,29(2):205-209.
[19] SERROR P,SASAKI T,EHRLICH S D,et al.Electrotransformation of Lactobacillus delbrueckii subsp.bulgaricus and L.delbrueckii subsp.lactis with various plasmids[J].Applied and Environmental Microbiology,2002,68(1):46-52.
[20] WEI M Q,RUSH C M,NORMAN J M,et al.An improved method for the transformation of Lactobacillus,strains using electroporation[J].Journal of Microbiological Methods,1995,21(1):97-109.
[2] SUN Z,LIU W,GAO W,et al.Identification and characterization of the dominant lactic acid bacteria from kurut:the naturally fermented yak milk in Qinghai,China[J].The Journal of General and Applied Microbiology,2010,56(1):1-10.
[3] LIU S N,HAN Y,ZHOU Z J.Lactic acid bacteria in traditional fermented Chinese foods[J].Food Research International,2011,44(3):643-651.
[4] LJUNGH A,WADSTR?M T.Lactic acid bacteria as probiotics[J].Current Issues In Microbiology,2006,7(2):73-89.
[5] KLAENHAMMER T R,ALTERMANN E,PFEILER E,et al.Functional genomics of probiotic Lactobacilli[J].Journal of Clinical Gastroenterology,2008,42(8):S160.
[6] 敖晓琳,蒲彪,蔡义民,等.发酵乳杆菌及其益生特性研究进展[J].食品与生物技术学报,2015(2):121-127.
[7] 王巧惠,王光强,宋馨,等.不同质粒电转三种乳酸菌的比较研究[J].工业微生物,2016,46(1):36-41.
[8] 凌代文,东秀珠.乳酸细菌分类鉴定及实验方法[J].微生物学通报,1998(1):23.
[9] EWE J A,WAN-ABDULLAH W N,ALIAS A K,et al.Bioconversion of isoflavones and the probiotic properties of the electroporated parent and subsequent three subcultures of Lactobacillus fermentum BT8219 in biotin-soymilk[J].Journal of Microbiology and Biotechnology,2012,22(22):947-959.
[10] RUSH C M,HAFNER L M,TIMMS P.Genetic modification of a vaginal strain of Lactobacillus fermentum and its maintenance within the reproductive tract after intravaginal administration[J].Journal of Medical Microbiology,1994,41(4):272.
[11] 郭兴华,曹郁生,东秀珠.益生乳酸细菌—分子生物学及生物技术[M].北京:科学出版社,2008.
[12] IRAM M,WANG X,ZHAO H Y,et al.Ameliorate maneuver for transformation of Lactobacillus strains by electroporation with IBDV-vp2 chemically engineered expression vector[J].Journal of Animal and Plant Sciences,2016,26(3):814-822.
[13] LUCHANSKY J B,MURIANA P M,KLAENHAMMER T R.Application of electroporation for transfer of plasmid DNA to Lactobacillus,Lactococcus,Leuconostoc,Listeria,Pediococcus,Bacillus,Staphylococcus,Enterococcus and Propionibacterium[J].Molecular Microbiology,1988,2(5):637-646.
[14] KIM Y H,HAN K S,OH S,et al.Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation[J].Journal of Applied Microbiology,2010,99(1):167-174.
[15] 张旭,崔艳华,张兰威,等.乳酸菌电转化条件研究[J].兰州大学学报(自科版),2009,45(s1):26-33.
[16] 曹晓梅,张虎成,李曼,等.乳杆菌电转化的研究进展[J].军事医学,2008,32(6):590-593.
[17] 万谦.乳酸菌质粒提取方法及电转化条件的研究[D].郑州:河南工业大学,2010.
[18] 夏子芳,石贵阳,张梁,等.乳杆菌电转化条件的优化[J].食品科学,2008,29(2):205-209.
[19] SERROR P,SASAKI T,EHRLICH S D,et al.Electrotransformation of Lactobacillus delbrueckii subsp.bulgaricus and L.delbrueckii subsp.lactis with various plasmids[J].Applied and Environmental Microbiology,2002,68(1):46-52.
[20] WEI M Q,RUSH C M,NORMAN J M,et al.An improved method for the transformation of Lactobacillus,strains using electroporation[J].Journal of Microbiological Methods,1995,21(1):97-109.