scholarly journals Scarless gene deletion using mazF as a new counter-selection marker and an improved deletion cassette assembly method in Saccharomyces cerevisiae

2014 ◽  
Vol 60 (2) ◽  
pp. 89-93 ◽  
Author(s):  
Quanli Liu ◽  
Huajuan Liu ◽  
Yanyan Yang ◽  
Xiuming Zhang ◽  
Yanling Bai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Deletion ◽  
Selection Marker ◽  
Assembly Method ◽  
Counter Selection ◽  
Deletion Cassette

Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method

Microbiology ◽  
2010 ◽  
Vol 156 (11) ◽  
pp. 3386-3397 ◽  
Author(s):  
Changyi Zhang ◽  
Li Guo ◽  
Ling Deng ◽  
Yuanxin Wu ◽  
Yunxiang Liang ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Target Gene ◽  
Gene Deletion ◽  
Nuclear Antigen ◽  
Pcr Analysis ◽  
Sulfolobus Islandicus ◽  
Counter Selection ◽  
Micro Organisms ◽  
Proliferating Cell ◽  
Mutant Cells

Organisms belonging to the Crenarchaeota lineage contain three proliferating cell nuclear antigen (PCNA) subunits, while those in the Euryarchaeota have only one, as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in Sulfolobus islandicus, a hyperthermophilic crenarchaeon, but failed with two conventional knockout methods. Then, a new knockout scheme, known as marker insertion and target gene deletion (MID), was developed, with which transformants were obtained for each pMID-pcna plasmid. We found that mutant cells persisted in transformant cultures during incubation of pMID-pcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semiquantitative PCR analysis of the deleted target gene allele (Δpcna1 or Δpcna3) revealed that mutant cells could no longer be propagated, demonstrating that these pcna genes are absolutely required for host cell viability. Because the only prerequisite for this assay is the generation of a MID transformant, this approach can be applied generally to any micro-organisms proficient in homologous recombination.

scholarly journals Capped mRNA Degradation Intermediates Accumulate in the Yeast spb8-2 Mutant

1998 ◽  
Vol 18 (9) ◽  
pp. 5062-5072 ◽  
Author(s):  
Ronald Boeck ◽  
Bruno Lapeyre ◽  
Christine E. Brown ◽  
Alan B. Sachs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Family Member ◽  
Gene Deletion ◽  
Binding Protein ◽  
Mrna Degradation ◽  
Protein Family ◽  
Suppressor Mutation ◽  
Mrna Decapping ◽  
Mrna Species ◽  
Degradation Intermediates

ABSTRACT mRNA in the yeast Saccharomyces cerevisiae is primarily degraded through a pathway that is stimulated by removal of the mRNA cap structure. Here we report that a mutation in the SPB8(YJL124c) gene, initially identified as a suppressor mutation of a poly(A)-binding protein (PAB1) gene deletion, stabilizes the mRNA cap structure. Specifically, we find that thespb8-2 mutation results in the accumulation of capped, poly(A)-deficient mRNAs. The presence of this mutation also allows for the detection of mRNA species trimmed from the 3′ end. These data show that this Sm-like protein family member is involved in the process of mRNA decapping, and they provide an example of 3′-5′ mRNA degradation intermediates in yeast.

Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms

2015 ◽  
Vol 11 (11) ◽  
pp. 3129-3136 ◽  
Author(s):  
Namal V. C. Coorey ◽  
James H. Matthews ◽  
David S. Bellows ◽  
Paul H. Atkinson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Mechanism Of Action ◽  
Gene Deletion ◽  
Genomic Library ◽  
Deletion Mutants ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA).

Comprehensive phenotypic analysis of single-gene deletion and overexpression strains of Saccharomyces cerevisiae

Yeast ◽  
2011 ◽  
Vol 28 (5) ◽  
pp. 349-361 ◽  
Author(s):  
Katsunori Yoshikawa ◽  
Tadamasa Tanaka ◽  
Yoshihiro Ida ◽  
Chikara Furusawa ◽  
Takashi Hirasawa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Deletion ◽  
Single Gene ◽  
Phenotypic Analysis ◽  
Single Gene Deletion

Revelation of molecular basis for chromium toxicity by phenotypes of Saccharomyces cerevisiae gene deletion mutants

Metallomics ◽  
2016 ◽  
Vol 8 (5) ◽  
pp. 542-550 ◽  
Author(s):  
Adam J. Johnson ◽  
Filip Veljanoski ◽  
Patrick. J. O'Doherty ◽  
Mohammad S. Zaman ◽  
Gayani Petersingham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Gene Deletion ◽  
Deletion Mutants ◽  
Chromium Toxicity

Development of a markerless gene replacement system in Corynebacterium glutamicum using upp as a counter-selection marker

2014 ◽  
Vol 37 (3) ◽  
pp. 609-617 ◽  
Author(s):  
Wenwen Ma ◽  
Xiaoyue Wang ◽  
Yufeng Mao ◽  
Zhiwen Wang ◽  
Tao Chen ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Gene Replacement ◽  
Selection Marker ◽  
Counter Selection ◽  
Replacement System

scholarly journals Development and optimization of Lysis gene E as a counter-selection marker with high selection stringency

2021 ◽  
Author(s):  
Wei Chen ◽  
Ruyi Chen ◽  
Ling He ◽  
Xiaotong Wu
Keyword(s):  
Regulatory System ◽  
Selection Marker ◽  
Marker Genes ◽  
Selection System ◽  
Gram Negative ◽  
E Coli ◽  
Lysis Gene ◽  
Counter Selection ◽  
Practical Research ◽  
High Selection

Seamless modification of bacteria chromosome is widely performed both in theoretical and in practical research, for this purpose, excellent counter-selection marker genes with high selection stringency are needed. Lysis gene E from bacteriophage PhiX174 was developed and optimized as a counter-selection marker in this paper. Lysis gene E was firstly constructed under the control of pL promoter. At 42 °C, Lysis gene E could effectively kill Escherichia coli. Seamless modification using E as a counter-selection marker also successfully conducted. It also works in another Gram-negative strain Serratia marcescens under the control of Arac/PBAD regulatory system. Through combining lysis gene E and kil, the selection stringency frequency of pL-kil-sd-E cassette in E. coli arrived at 4.9×10−8 and 3.2×10−8 at two test loci, which is very close to the best counter-selection system, inducible toxins system. Under the control of Arac/PBAD, selection stringency of PBAD-kil-sd-E in S. marcescens arrived at the level of 10−7 at four test loci. By introducing araC gene harboring plasmid pKDsg-ack, 5- to 18- fold improvement of selection stringency was observed at these loci, and a surprising low selection stringency frequency 4.9×10−9 was obtained at marR-1 locus, the lowest selection stringency frequency for counter-selection reported so far. Similarly, at araB locus of E. coli selection stringency frequency of PBAD-kil-sd-E was improved to 3×10−9 after introducing plasmid pKDsg-ack. In conclusion, we have developed and optimized a newly universal counter-selection marker based on lysis gene E. The best selection stringency of this new marker exceeds the inducible toxins system several fold.

scholarly journals GEDpm-cg: Genome Editing Automated Design Platform for Point Mutation Construction in Corynebacterium glutamicum

2021 ◽  
Vol 9 ◽  
Author(s):  
Yi Yang ◽  
Yufeng Mao ◽  
Ye Liu ◽  
Ruoyu Wang ◽  
Hui Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Point Mutation ◽  
In Silico ◽  
Computer Aided Design ◽  
Large Scale ◽  
Point Mutations ◽  
Dna Assembly ◽  
Cell Factories ◽  
Assembly Method ◽  
Counter Selection

Advances in robotic system-assisted genome editing techniques and computer-aided design tools have significantly facilitated the development of microbial cell factories. Although multiple separate software solutions are available for vector DNA assembly, genome editing, and verification, by far there is still a lack of complete tool which can provide a one-stop service for the entire genome modification process. This makes the design of numerous genetic modifications, especially the construction of mutations that require strictly precise genetic manipulation, a laborious, time-consuming and error-prone process. Here, we developed a free online tool called GEDpm-cg for the design of genomic point mutations in C. glutamicum. The suicide plasmid-mediated counter-selection point mutation editing method and the overlap-based DNA assembly method were selected to ensure the editability of any single nucleotide at any locus in the C. glutamicum chromosome. Primers required for both DNA assembly of the vector for genetic modification and sequencing verification were provided as design results to meet all the experimental needs. An in-silico design task of over 10,000 single point mutations can be completed in 5 min. Finally, three independent point mutations were successfully constructed in C. glutamicum guided by GEDpm-cg, which confirms that the in-silico design results could accurately and seamlessly be bridged with in vivo or in vitro experiments. We believe this platform will provide a user-friendly, powerful and flexible tool for large-scale mutation analysis in the industrial workhorse C. glutamicum via robotic/software-assisted systems.

scholarly journals Harnessing the Endogenous 2μ Plasmid of Saccharomyces cerevisiae for Pathway Construction

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Yang ◽  
Yujuan Tian ◽  
Huayi Liu ◽  
Yeyi Kan ◽  
Yi Zhou ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Manipulation ◽  
Marker Gene ◽  
Insertion Site ◽  
Selection Marker ◽  
Plasmid Copy ◽  
Genetic Manipulations ◽  
Copy Numbers ◽  
Improved Stability ◽  
Valuable Product

pRS episomal plasmids are widely used in Saccharomyces cerevisiae, owing to their easy genetic manipulations and high plasmid copy numbers (PCNs). Nevertheless, their broader application is hampered by the instability of the pRS plasmids. In this study, we designed an episomal plasmid based on the endogenous 2μ plasmid with both improved stability and increased PCN, naming it p2μM, a 2μ-modified plasmid. In the p2μM plasmid, an insertion site between the REP1 promoter and RAF1 promoter was identified, where the replication (ori) of Escherichia coli and a selection marker gene of S. cerevisiae were inserted. As a proof of concept, the tyrosol biosynthetic pathway was constructed in the p2μM plasmid and in a pRS plasmid (pRS423). As a result, the p2μM plasmid presented lower plasmid loss rate than that of pRS423. Furthermore, higher tyrosol titers were achieved in S. cerevisiae harboring p2μM plasmid carrying the tyrosol pathway-related genes. Our study provided an improved genetic manipulation tool in S. cerevisiae for metabolic engineering applications, which may be widely applied for valuable product biosynthesis in yeast.

scholarly journals Screening the Saccharomyces cerevisiae Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Kathy Parisi ◽  
Stephen R. Doyle ◽  
Eunice Lee ◽  
Rohan G. T. Lowe ◽  
Nicole L. van der Weerden ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Deletion ◽  
Screening Method ◽  
Large Family ◽  
Mechanisms Of Action ◽  
Sequencing Analysis ◽  
Differential Growth ◽  
Content Type ◽  
Plant Defensins ◽  
Nonessential Gene

ABSTRACT Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns. To gain a better understanding of the antifungal mechanisms of a set of four defensins, NaD1, DmAMP1, NbD6, and SBI6, we screened a pooled collection of the nonessential gene deletion set of Saccharomyces cerevisiae. Strains with increased or decreased ability to survive defensin treatment were identified based on the relative abundance of the strain-specific barcode as determined by MiSeq next-generation sequencing. Analysis of the functions of genes that are deleted in strains with differential growth in the presence of defensin provides insight into the mechanism of action. The screen identified a novel role for the vacuole in the mechanisms of action for defensins NbD6 and SBI6. The effect of these defensins on vacuoles was further confirmed by using confocal microscopy in both S. cerevisiae and the cereal pathogen Fusarium graminearum. These results demonstrate the utility of this screening method to identify novel mechanisms of action for plant defensins.

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