Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing

	Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing
	Zhang, Guo-qiang 1,3; Bao, Peng 1,2; Zhang, Yun 1; Deng, Ai-hua 1; Chen, Ning 2; Wen, Ting-yi 1
刊名	ANALYTICAL BIOCHEMISTRY
	2011-02-01
卷号	409 期号:1 页码:130-137
关键词	Recalcitrant Bacillus Electro-transformation Cell-wall weakening Cell-membrane fluidity disturbance
英文摘要	Bacillus amyloliquefaciens has been a major workhorse for the production of a variety of commercially important enzymes and metabolites for the past decades. Some subspecies of this bacterium are recalcitrant to exogenous DNA, and transformation with plasmid DNA is usually less efficient, thereby limiting the genetic manipulation of the recalcitrant species. In this work, a methodology based on electro-transformation has been developed, in which the cells were grown in a semicomplex hypertonic medium, cell walls were weakened by adding glycine (Gly) and DL-threonine (DL-Thr), and the cell-membrane fluidity was elevated by supplementing Tween 80. After optimization of the cell-loosening recipe by response surface methodology (RSM), the transformation efficiency reached 1.13 +/- 0.34 x 10(7) cfu/mu g syngeneic pUB110 DNA in a low conductivity electroporation buffer. Moreover, by temporary heat inactivation of the host restriction enzyme, a transformation efficiency of 8.94 +/- 0.77 x 10(5) cfu/mu g DNA was achieved with xenogeneic shuttle plasmids, a 10(3)-fold increase compared to that reported previously. The optimized protocol was also applicable to other recalcitrant B. amyloliquefaciens strains used in this study. This work could shed light on the functional genomics and subsequent strain improvement of the recalcitrant Bacillus, which are difficult to be transformed using conventional methods. (C) 2010 Elsevier Inc. All rights reserved.
WOS标题词	Science & Technology ; Life Sciences & Biomedicine ; Physical Sciences
类目[WOS]	Biochemical Research Methods ; Biochemistry & Molecular Biology ; Chemistry, Analytical
研究领域[WOS]	Biochemistry & Molecular Biology ; Chemistry
关键词[WOS]	CORYNEBACTERIUM-GLUTAMICUM ; ELECTROPORATION PROTOCOL ; NUCLEOTIDE-SEQUENCE ; PURINE NUCLEOSIDES ; CEREUS ATCC-14579 ; HIGH OSMOLARITY ; FUNCTIONAL MAP ; PLASMID DNA ; TRANSFORMATION ; SUBTILIS
收录类别	SCI
语种	英语
WOS记录号	WOS:000286126500018
内容类型	期刊论文
源URL	[http://124.16.173.210/handle/834782/1476]
专题	天津工业生物技术研究所_酶工程实验室马延和 _期刊论文
作者单位	1.Chinese Acad Sci, Inst Microbiol, Key Lab Syst Microbial Biotechnol, Beijing 100101, Peoples R China 2.Tianjin Univ Sci & Technol, Coll Biotechnol, Tianjin 300457, Peoples R China 3.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China
推荐引用方式 GB/T 7714	Zhang, Guo-qiang,Bao, Peng,Zhang, Yun,et al. Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing[J]. ANALYTICAL BIOCHEMISTRY,2011,409(1):130-137.
APA	Zhang, Guo-qiang,Bao, Peng,Zhang, Yun,Deng, Ai-hua,Chen, Ning,&Wen, Ting-yi.(2011).Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing.ANALYTICAL BIOCHEMISTRY,409(1),130-137.
MLA	Zhang, Guo-qiang,et al."Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing".ANALYTICAL BIOCHEMISTRY 409.1(2011):130-137.