scholarly journals pET expression vector customized for efficient seamless cloning

BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 384-387
Author(s):  
Dragana Dobrijevic ◽  
Lily A Nematollahi ◽  
Helen C Hailes ◽  
John M Ward
Keyword(s):  
Expression Vector ◽  
Gene Replacement ◽  
Expression Vectors ◽  
Gram Negative Bacteria ◽  
Golden Gate ◽  
Gram Negative ◽  
Large Numbers ◽  
Parallel Cloning ◽  
Natural Enzyme

Here we present a modification of the widely used pET29 expression vector for use in rapid and straightforward parallel cloning via a gene replacement and Golden Gate strategy. The modification can be applied to other expression vectors for Gram-negative bacteria. We have used the modified vectors to clone large numbers of bacterial natural enzyme variants from genomic and metagenomic sources for applications in biocatalysis.

scholarly journals Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a ΔbetCDE Pseudomonas putida Strain

2005 ◽  
Vol 71 (2) ◽  
pp. 883-892 ◽  
Author(s):  
Teca Calcagno Galvão ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
General Procedure ◽  
Gene Replacement ◽  
Selection Marker ◽  
Homologous Gene ◽  
Gram Negative Bacteria ◽  
Selection System ◽  
Gram Negative ◽  
Acid Sensitivity ◽  
Delivery Vector

ABSTRACT A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5′-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, where pyrF is associated with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-negative bacteria. The system has been instrumental for complete deletion of the P. putida choline-O-sulfate utilization operon betCDE, a mutant which could not be produced by any of the other genetic strategies available.

Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria

Plasmid ◽  
2007 ◽  
Vol 57 (3) ◽  
pp. 286-295 ◽  
Author(s):  
Attila Karsi ◽  
Mark L. Lawrence
Keyword(s):  
Host Range ◽  
Expression Vectors ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative

Increased Candida Demonstrated in Subgingival Plaques of Aids Patients by Microwave-Accelerated Silver Staining

1990 ◽  
Vol 189 ◽  
Author(s):  
Jacob S. Hanker ◽  
Mark J. Kutcher ◽  
E. Jefferson Burkes ◽  
George W. Greco ◽  
Roy L. Hopfer ◽  
...  
Keyword(s):  
Periodontal Disease ◽  
Oral Candidiasis ◽  
Periodic Acid ◽  
Rapid Test ◽  
Acid Oxidation ◽  
Subgingival Plaque ◽  
Gram Negative Bacteria ◽  
Aids Patients ◽  
Gram Negative ◽  
Large Numbers

ABSTRACTAcute periodontal lesions generally respond to conventional therapy. In AIDS patients, however, further deterioration is the usual consequence. Candida albicans(CA) lesions of the oral mucosa have been observed in 88% of AIDS patients; 59% of male homosexual and IV drug abuser oral candidiasis patients subsequently developed AIDS. Indeed HIV may be hard to find in AIDS patients in whom CA lesions are very prominent.Rapid reproducible silver stains have been developed in our laboratories to detect Gram-negative bacteria, fungi and protozoa. They demonstrate these microorganisms by the microwave-accelerated metallization of the aldehydes produced in their surface carbohydrates or lipopolysaccharides after periodic acid oxidation (Giammara and Hanker, US Patent No. 4,690,901, 1987; Sigma Diagnostics Kit No. HT-100). We thought that these stains might be useful to demonstrate by microscopy the large numbers of CA which culture studies had suggested were present in subgingival plaque at atypical periodontal disease sites of AIDS patients. Direct staining of plaque smears and their evaluation under the light microscope could be done in much less time than that required for culture. Any equivocal microorganisms could be positively identified as CA by immediate examination of the slide or coverslip by scanning electron microscopy. Although our PATS reaction was more valuable for identifying Gram-negative bacteria especially spirochetes and for studying substructure of CA and their epiphytic interactions with bacteria, the SIGMA DIAGNOSTICS Silver Stain HT-100 was much easier to use for rating CA concentration in disease sites. This test was applied to subgingival plaque smears from 28 periodontal disease sites of 22 HIV-seronegative patients and 45 sites of 12 HIV-seropositive patients. After staining, the smears were examined and scored (Davenport Index). None of the 12 HIVseropositive patients but 15 of the 22 HIV-seronegative patients had a score of zero at all sites. The score ranged from 0 (no CA) to 3 (large numbers of CA in all fields). In the seropositive patients the median score was 2 while in the seronegative group it was 0.The much higher scores obtained in specimens from periodontal disease sites of the AIDS patients suggest that this simple rapid test might be valuable for screening individuals and indicating those requiring further testing or monitoring for AIDS.

scholarly journals An improved version of suicide vector pKNG101 for gene replacement in Gram‐negative bacteria

1997 ◽  
Vol 23 (2) ◽  
pp. 410-411 ◽  
Author(s):  
Mahfuzur R. Sarker ◽  
Guy R. Cornelis
Keyword(s):  
Gene Replacement ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Suicide Vector

scholarly journals New Mobilizable Vectors Suitable for Gene Replacement in Gram-Negative Bacteria and Their Use in Mapping of the 3′ End of theXanthomonas campestris pv. campestrisgum Operon

1999 ◽  
Vol 65 (1) ◽  
pp. 278-282 ◽  
Author(s):  
Federico Katzen ◽  
Anke Becker ◽  
M. Verónica Ielmini ◽  
Cristian G. Oddo ◽  
Luis Ielpi
Keyword(s):  
Xanthomonas Campestris ◽  
Marker Gene ◽  
Gene Replacement ◽  
Gram Negative Bacteria ◽  
Double Crossover ◽  
Gram Negative ◽  
Marker Exchange

ABSTRACT We describe useful vectors to select double-crossover events directly in site-directed marker exchange mutagenesis in gram-negative bacteria. These vectors contain the gusA marker gene, providing colorimetric screens to identify bacteria harboring those sequences. The applicability of these vectors was shown by mapping the 3′ end of the Xanthomonas campestris gum operon, involved in biosynthesis of xanthan.

New Suicide Vector for Gene Replacement in Yersiniae and Other Gram-Negative Bacteria

Plasmid ◽  
1993 ◽  
Vol 29 (2) ◽  
pp. 160-163 ◽  
Author(s):  
Elzbieta Skrzypek ◽  
Pryce L. Haddix ◽  
Gregory V. Plano ◽  
Susan C. Straley
Keyword(s):  
Gene Replacement ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Suicide Vector

scholarly journals Gene Replacement in Gram-Negative Bacteria: the pMAKSAC Vectors

BioTechniques ◽  
2000 ◽  
Vol 28 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Didier Favre ◽  
Jean-François Viret
Keyword(s):  
Gene Replacement ◽  
Gram Negative Bacteria ◽  
Gram Negative

scholarly journals COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast

2019 ◽  
Author(s):  
Youlian Goulev ◽  
Audrey Matifas ◽  
Vincent Heyer ◽  
Bernardo Reina-San-Martin ◽  
Gilles Charvin
Keyword(s):  
Expression Vector ◽  
Experimental Approach ◽  
Target Location ◽  
Single Copy ◽  
Expression Vectors ◽  
Functional Modules ◽  
Golden Gate ◽  
Genetic Studies ◽  
Promoter Sequences ◽  
Expression Plasmids

AbstractA large number of genetic studies in yeast rely on the use of expression vectors. To facilitate the experimental approach of these studies, several collections of expression vectors have been generated (YXplac, pRS series, etc.). Subsequently, these collections have been expanded by adding more diversity to many of the plasmid features, including new selection markers and new promoter sequences. However, the ever growing number of plasmid feature makes it unrealistic for research labs to maintain an up-to-date collection of plasmids.. Here, we developed the COSPLAY toolbox: a Golden Gate approach based on the scheme of a simple modular plasmid that recapitulates and completes all the properties of the pRS plasmids. The COSPLAY toolbox contains a basal collection of individual functional modules. Moreover we standardized a simple and rapid, software-assisted protocol which facilitates the addition of new personalized modules. Finally, our toolbox includes the possibility to select a genomic target location and to perform a single copy integration of the expression vector.

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