A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii

Yeast ◽  
2019 ◽  
Vol 36 (12) ◽  
pp. 711-722
Author(s):  
Melissa Bizzarri ◽  
Stefano Cassanelli ◽  
Michala Dušková ◽  
Hana Sychrová ◽  
Lisa Solieri
Keyword(s):  
Antibiotic Resistance ◽  
Genetic Engineering ◽  
Cre Recombinase ◽  
Zygosaccharomyces Rouxii ◽  
Resistance Markers ◽  
Nonconventional Yeast

Centromeric DNA Facilitates Nonconventional Yeast Genetic Engineering

2017 ◽  
Vol 6 (8) ◽  
pp. 1545-1553 ◽  
Author(s):  
Mingfeng Cao ◽  
Meirong Gao ◽  
Carmen Lorena Lopez-Garcia ◽  
Yutong Wu ◽  
Arun Somwarpet Seetharam ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Centromeric Dna ◽  
Yeast Genetic ◽  
Nonconventional Yeast

Antibiotic resistance markers for plant transformation

1994 ◽  
pp. 125-137 ◽  
Author(s):  
Geert Angenon ◽  
Willy Dillen ◽  
Marc Van Montagu
Keyword(s):  
Antibiotic Resistance ◽  
Plant Transformation ◽  
Resistance Markers

scholarly journals Genome Engineering in Bacillus anthracis Using Cre Recombinase

2006 ◽  
Vol 74 (1) ◽  
pp. 682-693 ◽  
Author(s):  
Andrei P. Pomerantsev ◽  
Ramakrishnan Sitaraman ◽  
Craig R. Galloway ◽  
Violetta Kivovich ◽  
Stephen H. Leppla
Keyword(s):  
Antibiotic Resistance ◽  
Bacillus Anthracis ◽  
Genome Engineering ◽  
Genomic Sequence ◽  
Single Gene ◽  
Cre Recombinase ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Genomic Segment ◽  
Chromosomal Dna

ABSTRACT Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the complete genomic sequence of Bacillus anthracis, it is now possible to inactivate or delete selected genes of interest. However, many current methods for disrupting or deleting more than one gene require use of multiple antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic resistance marker into a selected region of the genome and subsequently removes it, leaving the target region (a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance cassette flanked by bacteriophage P1 loxP sites oriented as direct repeats was inserted within a selected gene. After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre recombinase action at the loxP sites excised the spectinomycin marker, leaving a single loxP site within the targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially mutated two pairs of genes: pepM and spo0A, and mcrB and mrr. Furthermore, loxP sites introduced at distant genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we deleted the capBCAD region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the mcrB and mrr genes, and in the latter case we found that the 32 intervening open reading frames were not essential to growth.

scholarly journals Bacteriophage 604: a marker phage for multi-resistantStaphylococcus aureusin Australia

1990 ◽  
Vol 104 (2) ◽  
pp. 211-218 ◽  
Author(s):  
B. Inglis ◽  
I. Heding ◽  
M. Merrylees ◽  
P. R. Stewart
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Chick Embryo ◽  
Molecular Marker ◽  
Resistance Markers

SUMMARYOf 28 multi-resistant isolates ofStaphylococcus aureuscollected during 1986 from hospitals in major cities around Australia, 27 were found to contain the same prophage (denoted phage 604). Hospital isolates carrying three or fewer resistance markers, and community isolates carrying one or no resistance markers, did not carry this prophage. Phage 604 does not confer antibiotic resistance on its lysogens, nor does it increase virulence in chick embryo assays. Phage 604 appears to be a correlate of antibiotic multi-resistance inS. aureusin Australia, and may provide a molecular marker for incipiently epidemic strains of this bacterium in Australian hospitals.

scholarly journals TnFLX: a third-generation mariner-based transposon system for Bacillus subtilis

2019 ◽  
Author(s):  
Felix Dempwolff ◽  
Sandra Sanchez ◽  
Daniel B. Kearns
Keyword(s):  
Antibiotic Resistance ◽  
Number System ◽  
Cellular Biology ◽  
Host Organism ◽  
E Coli ◽  
Genetic Tool ◽  
Translational Activity ◽  
Vector Construction ◽  
Low Copy Number ◽  
Resistance Markers

AbstractRandom transposon mutagenesis is a powerful genetic tool to answer fundamental biological questions in an unbiased approach. Here, we introduce an improved mariner-based transposon system with higher stability, and with versatile applications. We take advantage of the lower frequency of unintended recombination during vector construction and propagation in a low copy number system in E. coli to improve construct integrity. We generated a variety of transposons allowing for gene disruption or artificial overexpression each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance flexibility of future transposon modification and application in Bacillus and other organisms.ImportanceThe optimization of transposase encoding vectors in terms of stability during cloning and propagation is crucial for the reliable application of this system in any host organism. With an increased number of antibiotic resistance markers and the possibility to detect translational activity, the TnFLX transposon system will significantly help the implication of forward genetic methods in the field of cellular biology.

scholarly journals A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi.

2020 ◽  
Author(s):  
Constantin N. Takacs ◽  
Molly Scott ◽  
Yunjie Chang ◽  
Zachary A. Kloos ◽  
Irnov Irnov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antibiotic Resistance ◽  
Borrelia Burgdorferi ◽  
Gene Function ◽  
Electron Tomography ◽  
Ease Of Use ◽  
Fast Method ◽  
Cellular Motility ◽  
Crispr Interference ◽  
Resistance Markers

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi. For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi. Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen. IMPORTANCE Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

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