Genetic Manipulation System in Propionibacteria.

2002 ◽  
Vol 93 (1) ◽  
pp. 1-8 ◽  
Author(s):  
PORNPIMON KIATPAPAN ◽  
YOSHIKATSU MUROOKA
Keyword(s):  
Genetic Manipulation ◽  
Manipulation System

scholarly journals Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas

2015 ◽  
Vol 14 (1) ◽  
pp. 11 ◽  
Author(s):  
Pengxia Wang ◽  
Zichao Yu ◽  
Baiyuan Li ◽  
Xingsheng Cai ◽  
Zhenshun Zeng ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Manipulation System

scholarly journals Genetic Manipulation of a Lipolytic Yeast Candida aaseri SH14 Using CRISPR-Cas9 System

2020 ◽  
Vol 8 (4) ◽  
pp. 526 ◽  
Author(s):  
Zool Hilmi Ibrahim ◽  
Jung-Hoon Bae ◽  
Sun-Hee Lee ◽  
Bong Hyun Sung ◽  
Ahmad Hazri Ab Rashid ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Genetic Manipulation ◽  
Plant Oils ◽  
Long Chain Fatty Acids ◽  
Temporal Expression ◽  
Autonomously Replicating Sequence ◽  
Manipulation System ◽  
Episomal Vectors ◽  
Dodecanedioic Acid ◽  
Diploid Cells

A lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A β-oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of β-oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.

Genetic manipulation system in propionibacteria

2002 ◽  
Vol 93 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Pornpimon Kiatpapan ◽  
Yoshikatsu Murooka
Keyword(s):  
Genetic Manipulation ◽  
Manipulation System

scholarly journals An Efficient Genetic Manipulation Protocol for Dark Septate Endophyte Falciphora Oryzae

2021 ◽  
Author(s):  
Zhen-Zhu Su ◽  
Meng-Di Dai ◽  
Jia-Nan Zhu ◽  
Yu-Lan Zeng ◽  
Xuan-Jun Lu ◽  
...  
Keyword(s):  
Endophytic Fungus ◽  
Plant Biomass ◽  
Genetic Manipulation ◽  
Blast Resistance ◽  
Enzyme Digestion ◽  
Dark Septate Endophyte ◽  
Rice Blast Resistance ◽  
Rice Roots ◽  
Manipulation System ◽  
Protoplast Preparation

Abstract Falciphora oryzae is a dark septate endophyte (DSE) isolated from wild rice roots (Oryza sativa L.). It was classified as a non-clavicitaceous endophyte. The fungus colonizes rice roots, showing a significant increase in agronomic parameters with plant biomass, rice blast resistance, yield, and quality. The construction of the genetic manipulation system is critical to study the relationship between F. oryzae and O. sativa. In the present study, the protoplast preparation and transformation system of F. oryzae was investigated. The key parameters affecting the efficiency of protoplast production, such as osmotic pressure stabilizer, enzyme digestion conditions, and fungal age, were studied. The results showed that F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9mol L-1 KCl solution and 10 mg mL−1 glucanase at 30℃ with shaking 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar (RgA) medium containing 1M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired GFP-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots.The genetic manipulation system of endophytic fungus facilitates the further exploration the interaction between the endophytic fungus and their hosts.

Development of a genetic manipulation system for Haemophilus parasuis

2005 ◽  
Vol 105 (3-4) ◽  
pp. 223-228 ◽  
Author(s):  
Anna Bigas ◽  
M. Elena Garrido ◽  
Ana M. Pérez de Rozas ◽  
Ignacio Badiola ◽  
Jordi Barbé ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Haemophilus Parasuis ◽  
Manipulation System

Using 16S rDNA as Target Site for Homologous Recombination to Improve the Alkaline Protease Production of Bacillus alcalophilus

2014 ◽  
Vol 886 ◽  
pp. 349-354
Author(s):  
Qing Shan Mo ◽  
Yao Tian ◽  
Hui Tu Zhang ◽  
Ling Jun Bu ◽  
Fu Ping Lu
Keyword(s):  
16S Rdna ◽  
Alkaline Protease ◽  
Genetic Manipulation ◽  
Protease Production ◽  
Protease Gene ◽  
Wild Type ◽  
Recombinant Strains ◽  
Manipulation System ◽  
Rdna Sites ◽  
Alkaline Protease Gene

Bacillus alcalophilusisolated was used for the production of alkaline protease. The enzyme encoded by alkaline protease gene (apr4) gene. To further improve the production of the strain for industrial requirement, a genetic manipulation system forBacillus alcalophiluswas developed. Additional copies of theapr4 gene were transferred into the strainBacillus alcalophilusand integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated K23, exhibited superior properties for production of alkaline protease. the protease activity of K23 achieved by (6.19 ± 0.34) × 104U/ml, which is approximately 111.3% higher than that of the wild-type ones for 50-h fermentation. In addition, the new strain was genetically stable for more than 100 generations. These superior characteristics make it to be more suitable than the wild-type strain for alkaline protease production.

scholarly journals Isolation and Molecular Identification of Auxotrophic Mutants to Develop a Genetic Manipulation System for the HaloarchaeonNatrinemasp. J7-2

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Jie Lv ◽  
Shuai Wang ◽  
Yuchen Wang ◽  
Yuping Huang ◽  
Xiangdong Chen
Keyword(s):  
Genomic Dna ◽  
Genetic Manipulation ◽  
Gene Mutations ◽  
Transformation Method ◽  
Wild Type ◽  
Auxotrophic Mutants ◽  
Archaeal Species ◽  
Manipulation System ◽  
Exogenous Genes ◽  
Mutant Phenotypes

Our understanding of the genusNatrinemais presently limited due to the lack of available genetic tools. Auxotrophic markers have been widely used to construct genetic systems in bacteria and eukaryotes and in some archaeal species. Here, we isolated four auxotrophic mutants ofNatrinemasp. J7-2, via 1-methyl-3-nitro-1-nitroso-guanidin mutagenesis, and designated them as J7-2-1, J7-2-22, J7-2-26, and J7-2-52, respectively. The mutant phenotypes were determined to be auxotrophic for leucine (J7-2-1), arginine (J7-2-22 and J7-2-52), and lysine (J7-2-26). The complete genome and the biosynthetic pathways of amino acids in J7-2 identified that the auxotrophic phenotype of three mutants was due to gene mutations inleuB(J7-2-1),dapD(J7-2-26), andargC(J7-2-52). These auxotrophic phenotypes were employed as selectable makers to establish a transformation method. The transformation efficiencies were determined to be approximately 103transformants perµg DNA. And strains J7-2-1 and J7-2-26 were transformed into prototrophic strains with the wild type genomic DNA, amplified fragments of the corresponding genes, or the integrative plasmids carrying the corresponding genes. Additionally, exogenous genes,bgaHoramyHgene, were expressed successfully in J7-2-1. Thus, we have developed a genetic manipulation system for theNatrinemagenus based on the isolated auxotrophic mutants ofNatrinemasp. J7-2.

scholarly journals Developing a broadly applicable pyrF-based genetic manipulation system in Acinetobacter baumannii

2020 ◽  
Author(s):  
Run Xu ◽  
Can Gao ◽  
Shuqi Wu ◽  
Mengjiao Su ◽  
Chengfu Sun ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Gene Editing ◽  
Selectable Marker ◽  
Genetic Manipulation ◽  
Mortality Rates ◽  
Drug Resistant ◽  
Flanking Sequence ◽  
Time Saving ◽  
Counter Selection ◽  
Manipulation System

AbstractAcinetobacter baumannii is an emergency pathogenic bacterium for its multidrug-resistance and high mortality rates after infection. In-depth genetic analysis of A. baumannii virulence and drug-resistant genes is highly desirable. Existing methods for genetic manipulation of A. baumannii mainly rely on the use of antibiotic as the selectable marker, and the sacB/sucrose as the counter-selectable marker, which is inconvenient and inappropriate for all research of A. baumannii. Based on the highly conserved pyrF gene and its conserved 500bp-flanking sequence, we quickly and easily generated the pyrF-deleted mutants as the uracil auxotrophic host strain in three model strains and 11 clinical strains. The pyrF-carried vectors constructed for gene editing with pyrF/5-FOA as the counter-selection were conveniently and time-saving in these pyrF-deleted mutants. Utilizing the pyrF-based genetic manipulation system, we easily and efficiently modified the cas gene and CRISPR sequence of I-F CRISPR-Cas system in A. baumannii AYE, and detected the CRISPR interference and adaptation in these mutants. In summary, the pyrF-based genetic manipulation system could be broadly applicable used for efficiently maker-less gene editing in most A. baumannii strains.

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