scholarly journals Shuttle Vector-Based Transformation System for Pyrococcus furiosus

2010 ◽  
Vol 76 (10) ◽  
pp. 3308-3313 ◽  
Author(s):  
Ingrid Waege ◽  
Georg Schmid ◽  
Sybille Thumann ◽  
Michael Thomm ◽  
Winfried Hausner
Keyword(s):  
Pyrococcus Furiosus ◽  
Shuttle Vector ◽  
Gel Filtration ◽  
Model Organism ◽  
Vector System ◽  
Transformation System ◽  
Reductase Gene ◽  
Genetic Transformation System ◽  
Coa Reductase ◽  
Pyrococcus Abyssi

ABSTRACT Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi. In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus, allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furio s us, but the copy number was only one to two per chromosome. This system was used for overexpression of His6-tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter.

scholarly journals Establishment of a Tractable Genetic Transformation System in Veillonella spp.

2012 ◽  
Vol 78 (9) ◽  
pp. 3488-3491 ◽  
Author(s):  
Jinman Liu ◽  
Zhoujie Xie ◽  
Justin Merritt ◽  
Fengxia Qi
Keyword(s):  
Escherichia Coli ◽  
Genetic Transformation ◽  
Shuttle Vector ◽  
Transformation System ◽  
Genetic Studies ◽  
Content Type ◽  
Genetic Transformation System

ABSTRACTWe have constructed the firstEscherichia coli-Veillonellashuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinicalVeillonellastrain. A highly transformableVeillonellastrain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for futureVeillonellagenetic studies.

scholarly journals Construction of a Shuttle Vector for, and Spheroplast Transformation of, the Hyperthermophilic Archaeon Pyrococcus abyssi

2002 ◽  
Vol 68 (11) ◽  
pp. 5528-5536 ◽  
Author(s):  
Soizick Lucas ◽  
Laurent Toffin ◽  
Yvan Zivanovic ◽  
Daniel Charlier ◽  
Hélène Moussard ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Low Frequency ◽  
Vector System ◽  
Hyperthermophilic Archaea ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon ◽  
Shuttle Vectors ◽  
Bacterial Vector ◽  
Genes Encoding ◽  
Pyrococcus Abyssi

ABSTRACT Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (102 to 103 transformants per μg of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

scholarly journals Standardized Genetic Transformation Protocol for Chrysanthemum cv. ‘Jinba’ with TERMINAL FLOWER 1 Homolog CmTFL1a

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 860
Author(s):  
Saba Haider ◽  
Yaohui Gao ◽  
Yike Gao
Keyword(s):  
Transgenic Plants ◽  
Genetic Transformation ◽  
Model Organism ◽  
Plant Morphology ◽  
Pcr Analysis ◽  
Transformation System ◽  
Transformation Protocol ◽  
Genetic Transformation System ◽  
Chrysanthemum X Morifolium

Chrysanthemum (Chrysanthemum x morifolium Ramat.) cultivar Jinba is a distinctive short-day chrysanthemum that can be exploited as a model organism for studying the molecular mechanism of flowering. The commercial value of Jinba can be increased in global flower markets by developing its proper regeneration and genetic transformation system. By addressing typical problems associated with Agrobacterium-mediated transformation in chrysanthemum, that is, low transformation efficiency and high cultivar specificity, we designed an efficient, stable transformation system. Here, we identify the features that significantly affect the genetic transformation of Jinba and standardize its transformation protocol by using CmTFL1a as a transgene. The appropriate concentrations of various antibiotics (kanamycin, meropenem and carbenicillin) and growth regulators (6-BA, 2,4-D and NAA) for the genetic transformation were determined to check their effects on in vitro plant regeneration from leaf segments of Jinba; thus, the transformation protocol was standardized through Agrobacterium tumefaciens (EHA105). In addition, the presence of the transgene and its stable expression in CmTFL1a transgenic plants were confirmed by polymerase chain reaction (PCR) analysis. The CmTFL1a transgene constitutively expressed in the transgenic plants was highly expressed in shoot apices as compared to stem and leaves. Overexpression of CmTFL1a led to a delay in transition to the reproductive phase and significantly affected plant morphology. This study will help to understand the biological phenomenon of TFL1 homolog in chrysanthemum. Moreover, our findings can explore innovative possibilities for genetic engineering and breeding of other chrysanthemum cultivars.

scholarly journals Utilization of Virus ϕCh1 Elements To Establish a Shuttle Vector System for Halo(alkali)philic Archaea via Transformation of Natrialba magadii

2013 ◽  
Vol 79 (8) ◽  
pp. 2741-2748 ◽  
Author(s):  
M. Mayrhofer-Iro ◽  
A. Ladurner ◽  
C. Meissner ◽  
C. Derntl ◽  
M. Reiter ◽  
...  
Keyword(s):  
Heterologous Gene Expression ◽  
Shuttle Vector ◽  
Extreme Environments ◽  
Vector System ◽  
Structural Protein ◽  
Transformation System ◽  
Content Type ◽  
Shuttle Vectors ◽  
Modified Method ◽  
Natrialba Magadii

ABSTRACTIn the study described here, we successfully developed a transformation system for halo(alkali)philic members of theArchaea. This transformation system comprises a series ofNatrialba magadii/Escherichia colishuttle vectors based on a modified method to transform halophilic members of theArchaeaand genomic elements of theN. magadiivirus ϕCh1. The shuttle vector pRo-5, based on therepH-containing region of ϕCh1, stably replicated inE. coliandN. magadiiand in several halophilic and haloalkaliphilic members of theArchaeanot transformable so far. The ϕCh1 operon ORF53/ORF54 (repH) was essential for pRo-5 replication and was thus identified as the minimal replication origin. The plasmid allowed homologous and heterologous gene expression, as exemplified by the expression of ϕCh1 ORF3452, which encodes a structural protein, and the reporter genebgaHofHaloferax lucentenseinN. magadii. The new transformation/vector system will facilitate genetic studies withinN. magadiiand other haloalkaliphilic archaea and will allow the detailed characterization of the gene functions ofN. magadiivirus ϕCh1 in their extreme environments.

scholarly journals Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

2021 ◽  
Vol 7 (2) ◽  
pp. 138
Author(s):  
Min Liang ◽  
Wei Li ◽  
Landa Qi ◽  
Guocan Chen ◽  
Lei Cai ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Genetic Manipulation ◽  
Major Facilitator Superfamily ◽  
Protoplast Regeneration ◽  
Transformation System ◽  
Regeneration Rate ◽  
Bioactive Natural Products ◽  
Genetics Research ◽  
Genetic Transformation System ◽  
Major Facilitator

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

scholarly journals Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

2002 ◽  
Vol 68 (12) ◽  
pp. 6237-6245 ◽  
Author(s):  
Tara D. Sutherland ◽  
Irene Horne ◽  
Robyn J. Russell ◽  
John G. Oakeshott
Keyword(s):  
Shuttle Vector ◽  
Open Reading Frame ◽  
Cosmid Library ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Reading Frame ◽  
Reductase Gene ◽  
Degradation Activity ◽  
Layer Chromatography

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

Establishment of efficient callus genetic transformation system for Pyrus armeniacaefolia

2021 ◽  
Vol 289 ◽  
pp. 110429
Author(s):  
Xinhui Wang ◽  
Fengli Zhou ◽  
Jianlong Liu ◽  
Wenqian Liu ◽  
Shaoling Zhang ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Transformation System ◽  
Genetic Transformation System

Efficient plant regeneration and genetic transformation system of the precious fast-growing tree Toona ciliata

2021 ◽  
Vol 172 ◽  
pp. 114015
Author(s):  
Wenmai Mao ◽  
Huiyun Song ◽  
Yue Li ◽  
Yueyang Wang ◽  
Huijuan Lin ◽  
...  
Keyword(s):  
Plant Regeneration ◽  
Genetic Transformation ◽  
Transformation System ◽  
Fast Growing ◽  
Toona Ciliata ◽  
Genetic Transformation System
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