恶臭假单胞菌KT2440高效电转化方法

Highly Efficient Transformation of Pseudomonas putida KT2440 by Electroporation

恶臭假单胞菌(Pseudomonas putida)KT2440是模式环境微生物菌株,也是异源表达的良好宿主菌.高效率的电转化方法对于基因的引入及后续实验非常关键.通过条件优化,建立了高效的电转化方法.EM培养基培养菌体至D600nm=0.6～0.75,冰冷的3mmol/L4-羟乙基哌嗪乙磺酸(HEPES,pH=7.0)的缓冲液洗涤菌体3次,菌体浓缩300倍,50μL分装并用于一次电转化.电转化条件:1.2kV,200Ω,25μF.1mL SOC培养基悬浮后,转至15mL聚丙烯离心管培养2h,转化效率可高达3.0×108转化子/μg,比目前为止文献报道的该菌株电转化效率高出30倍左右.实验结果还表明,转化DNA浓度与转化子数目呈线性关系.

Pseudomonas putida KT2440 is an archetype environmental microbe and an excellent host for heterologous gene expression. Highly efficient DNA transformation facilitates gene introduction and follow-up experiments. In this study, the electroporation conditions of P. putida KT2440 were optimized, and EM was used as liquid culture medium. When optical culture density at D600 nm reached 0.60-0.75, it was pelleted at 4℃ and washed three times with ice-cold 3 mmol/L HEPES （pH=7.0）, and finally suspended in the same buffer with 1/300 condensation. 50μL was used for one electroporation using Bio-Rad Gene-Pulser II with settings at 1.2 kV, 200 12 and 25μF, after which 1 mL SOC was added and the suspension was transferred to 15 mL polypropylene tube for 2 hours incubation at 30℃. With pCM132 and pJB861 as incoming DNA, transformation efficiency could be up to 3.0×10^8/μg, which was 30 times as high as the reference reported. Linear correlation was observed between DNA amount and the number of transformants. Fig 2, Tab 1, Ref 14