1 Introduction Haloarchaea represents a distinct group of Archaea that typically inhabits hypersaline environments,such as salt lakes and sea salterns.They are easy to culture and many haloarchaea are genetically trac...1 Introduction Haloarchaea represents a distinct group of Archaea that typically inhabits hypersaline environments,such as salt lakes and sea salterns.They are easy to culture and many haloarchaea are genetically tractable,hence they are excellent model systems for research of archaeal genetics,展开更多
The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their ...The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon （apyrF） as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding apyrF host. After positive selection of the single-crossover integration recombinants （pop-in） on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants （pop-out） with 5-fluoroorotic acid （5-FOA）, the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.展开更多
[Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea.For gene expression and function analysis of specific genes in model strain Haloarcula hispa...[Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea.For gene expression and function analysis of specific genes in model strain Haloarcula hispanica,a novel shuttle expression vector based on pSCM201 was developed.[Methods] By combining the minimal replicon of pSCM201,the pUC19 replicon of Escherichia coli,and two antibiotic genes,a new shuttle vector between haloarchaea and E.coli was established.Furthermore,the hsp5 core promoter,multiple clone sites and a C-terminal His·Tag sequence were added to construct the expression vector pSCM307.With the help of reporter gene bgaH,the replication ability and gene expression of pSCM307 were tested in H.hispanica AS2049 by PCR identification and β-galactosidase activity assay.[Results] pSCM307 could stably self-replicate in AS2049,and bgaH was successfully expressed under the control of hsp5 promoter.[Conclusions] A user-friendly expression vector was successfully constructed in haloarchaea.展开更多
基金supported by grants from the National 863 Program of Chinathe National Natural Science Foundation of Chinathe Chinese Academy of Sciences
文摘1 Introduction Haloarchaea represents a distinct group of Archaea that typically inhabits hypersaline environments,such as salt lakes and sea salterns.They are easy to culture and many haloarchaea are genetically tractable,hence they are excellent model systems for research of archaeal genetics,
基金supported by the National Natural Science Foundation of China(Nos.30830004,30925001)the National 863 Program of China(No.2009AA09Z401)the Chinese Academy of Sciences(No.KSCX2-EW-G-2)
文摘The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon （apyrF） as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding apyrF host. After positive selection of the single-crossover integration recombinants （pop-in） on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants （pop-out） with 5-fluoroorotic acid （5-FOA）, the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.
文摘[Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea.For gene expression and function analysis of specific genes in model strain Haloarcula hispanica,a novel shuttle expression vector based on pSCM201 was developed.[Methods] By combining the minimal replicon of pSCM201,the pUC19 replicon of Escherichia coli,and two antibiotic genes,a new shuttle vector between haloarchaea and E.coli was established.Furthermore,the hsp5 core promoter,multiple clone sites and a C-terminal His·Tag sequence were added to construct the expression vector pSCM307.With the help of reporter gene bgaH,the replication ability and gene expression of pSCM307 were tested in H.hispanica AS2049 by PCR identification and β-galactosidase activity assay.[Results] pSCM307 could stably self-replicate in AS2049,and bgaH was successfully expressed under the control of hsp5 promoter.[Conclusions] A user-friendly expression vector was successfully constructed in haloarchaea.
