Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants

doi:10.1007/s00253-013-4837-6

QIBEBT-IR > 微生物代谢工程研究组

	Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants
	Tan, Xiaoming 1; Liang, Feiyan1,2 ; Cai, Ke 1; Lu, Xuefeng 1
	2013-07-01
发表期刊	APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
ISSN	0175-7598
卷号	97 期号:14 页码:6373-6382
摘要	Due to efficient photosynthetic capability, robust growth, and clear genetic background, cyanobacteria are recently used for production of different biofuel and biochemical molecules by genetic engineering and showed great potentials as the next-generation microbial cell factory. For improving the production of bio-products, a number of genetic modifications are important for cyanobacteria. However, the system-level genetic modification of cyanobacteria is limited by the lack of efficient method for marker recycling. In this investigation, we introduced the self-replicable shutter vectors harboring the flipase (FLP) gene from Saccharomyces cerevisiae into two mutants of Synechocystis sp. PCC6803 and Synechococcus elongatus PCC7942 whose genomes were inserted by a kanamycin resistance gene with flipase recombination target (FRT) flanking, respectively. Transcriptional analysis by reverse transcription polymerase chain reaction showed that FLP gene was transcripted in both the two cyanobacterial strains. Genotyping analysis indicated that FLP performed its function in vivo in both two cyanobacterial strains, and the following DNA sequencing analysis on the targeted loci further confirmed that FLP exactly excised and ligated the two FRT sites between which a kanamycin resistance gene is located. The homozygous mutants free of the kanamycin resistance gene cassette were obtained by conditional expression of FLP and further dilution plating. The shuttle vectors carrying the FLP gene were then lost in these mutants by growing in the absence of antibiotics and the further single colony separation. These results demonstrate that FLP/FRT recombination system is able to be applied to the construction of markerless mutant in both Synechocystis sp. PCC6803 and S. elongatus PCC7942.
文章类型	Article
关键词	Flp/frt Cyanobacteria Markerless Mutant Marker Recycling Genetic Engineering Biofuels
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
DOI	10.1007/s00253-013-4837-6
关键词[WOS]	CARBON-DIOXIDE ; PHOTOSYNTHETIC PRODUCTION ; ESCHERICHIA-COLI ; PCC 6803 ; CONVERSION ; SELECTION ; GENE ; BIOTECHNOLOGY ; PCC-7120 ; ETHYLENE
收录类别	SCI
语种	英语
WOS研究方向	Biotechnology & Applied Microbiology
项目资助者	National Basic Research Program of China(973: 2011CBA00907) ; Knowledge Innovation Program of the Chinese Academy of Sciences(KSCX2-EW-G-1-4) ; Youth Innovation Promotion Association(Y21A01110A) ; "100-Talent Program of the Chinese Academy of Sciences" foundation(O91001110A)
WOS类目	Biotechnology & Applied Microbiology
WOS记录号	WOS:000321121800024
出版者	SPRINGER
引用统计	被引频次：28[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/1638
专题	微生物代谢工程研究组
通讯作者	Lu, Xuefeng
作者单位	1.Chinese Acad Sci, Shandong Prov Key Lab Energy Genet, Qingdao Inst Bioenergy & Bioproc Technol, Key Lab Biofuels, Qingdao 266101, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
推荐引用方式 GB/T 7714	Tan, Xiaoming,Liang, Feiyan,Cai, Ke,et al. Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants[J]. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY,2013,97(14):6373-6382.
APA	Tan, Xiaoming,Liang, Feiyan,Cai, Ke,&Lu, Xuefeng.(2013).Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants.APPLIED MICROBIOLOGY AND BIOTECHNOLOGY,97(14),6373-6382.
MLA	Tan, Xiaoming,et al."Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants".APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 97.14(2013):6373-6382.

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