Efficient Electrotransformation of Bacteroides fragilis
Efficient Electrotransformation of Bacteroides fragilis
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DOI：
10.1128/aem.02420-09
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发表时间：
2010-05-01
影响因子：
4.4
通讯作者：
Kuwahara, Tomomi
中科院分区：
文献类型：
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作者：
Ichimura, Minoru;Nakayama-Imaohji, Haruyuki;Kuwahara, Tomomi
本研究描述了改进的电穿孔参数，有效地转化脆弱拟杆菌的质粒制备的实验室菌株的大肠杆菌。所用方法的开发包括确定感受态细胞制备的最佳生长条件、可选择的抗菌素耐药性标记、电场强度和脉冲后孵育时间。四个E。大肠杆菌-拟杆菌穿梭质粒（pVAL-1、pVAL-2、pNLY 1和pLYL 05）中，发现含有头孢西丁抗性标记的pLYL 05最适合于B。fragilis转化，并且它产生的转化体（约10（4）个转化体/μ g pLYL 05 DNA）比其它质粒多2- 900倍。在72 h培养期内，B.在48小时收获的fragilis细胞产生最高数量的转化体。在5.0 ~ 12.5kV/cm范围内，pLYL 05的转化效率与电场强度呈线性关系。至少需要3小时的脉冲后孵育，以最大限度地提高转化效率。删除B。通过同源重组将pLYL 05自杀载体与fragilis基因进行同源重组，需要生长到早期指数期的感受态细胞和12小时的脉冲后孵育，以将基于pLYL 05的自杀载体有效整合到靶位点中。在B中获得了预期的积分。仅当同源制备（即，体内甲基化）自杀载体。自发的二倍体解析成功地删除了预期的遗传区域。我们的简单而有效的质粒转移方法能够破坏一个B。fragilis基因，使用体内甲基化的靶向载体。我们优化的电穿孔参数为拟杆菌属物种的遗传操作提供了有用的工具。
This study describes refined electroporation parameters for efficient transformation of Bacteroides fragilis by plasmids prepared from laboratory strains of Escherichia coli. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four E. coli-Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable for B. fragilis transformation, and it generated 2- to 900-fold more transformants (about 10(4) transformants per mu g pLYL05 DNA) than the other plasmids. For the 72-h cultivation period tested, B. fragilis cells harvested at 48 h yielded the highest numbers of transformants. The transformation efficiency of pLYL05 increased linearly with the electric field strength over a range from 5.0 to 12.5 kV/cm. At least 3 h of postpulse incubation was required to maximize the transformation efficiency. For deletion of B. fragilis genes by homologous recombination, competent cells grown to early exponential phase and 12 h of postpulse incubation were required for efficient integration of the pLYL05-based suicide vector into the target site. The expected integration was obtained in B. fragilis strain NCTC9343 only when a homologously prepared (i.e., in vivo methylated) suicide vector was used. Spontaneous resolution of the diploid successfully deleted the expected genetic region. Our simple and efficient plasmid transfer method enabled disruption of a B. fragilis gene using in vivo-methylated targeted vectors. Our optimized electroporation parameters provide a useful tool for genetic manipulation of Bacteroides species.