scholarly journals Free fatty acids promote transformation efficiency of yeast

2019 ◽  
Vol 19 (7) ◽  
Author(s):  
Xingpeng Duan ◽  
Xiaojing Ma ◽  
Shengying Li ◽  
Yongjin J Zhou
Keyword(s):  
Transformation Efficiency ◽  
Culture Media ◽  
Cell Factory ◽  
Competent Cell ◽  
Cell Preparation ◽  
Yeast Transformation ◽  
High Transformation Efficiency ◽  
Metabolic Analysis ◽  
High Transformation ◽  
Large Plasmids

ABSTRACT High transformation efficiency is essential in genetic engineering for functional metabolic analysis and cell factory construction, in particular in construction of long biosynthetic pathways with multiple genes. Here, we found that free fatty acid (FFA)-overproducing strain showed higher transformation efficiency in Saccharomyces cerevisiae. We then verified that external supplementation of FFAs, to the culture media for competent cell preparation, improved yeast transformation efficiency significantly. Among all tested FFAs, 0.5 g/L C16:0 FFA worked best on promoting transformation of S. cerevisiae and Komagataella phaffii (previously named as Pichia pastoris). Furthermore, C16:0 FFA improved the assembly efficiency of multiple DNA fragments into large plasmids and genome by 100%, which will facilitate the construction and optimization of multigene-containing long pathways.

scholarly journals Exploration of an Efficient Electroporation System for Heterologous Gene Expression in the Genome of Methanotroph

2021 ◽  
Vol 12 ◽  
Author(s):  
Lizhen Hu ◽  
Shuqi Guo ◽  
Xin Yan ◽  
Tianqing Zhang ◽  
Jing Xiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transformation Efficiency ◽  
Heterologous Gene Expression ◽  
Low Cost ◽  
Heterologous Gene ◽  
Industrial Biotechnology ◽  
Cell Factory ◽  
Competent Cell ◽  
High Transformation Efficiency ◽  
High Transformation

One-carbon (C1) substrates such as methane and methanol have been considered as the next-generation carbon source in industrial biotechnology with the characteristics of low cost, availability, and bioconvertibility. Recently, methanotrophic bacteria naturally capable of converting C1 substrates have drawn attractive attention for their promising applications in C1-based biomanufacturing for the production of chemicals or fuels. Although genetic tools have been explored for metabolically engineered methanotroph construction, there is still a lack of efficient methods for heterologous gene expression in methanotrophs. Here, a rapid and efficient electroporation method with a high transformation efficiency was developed for a robust methanotroph of Methylomicrobium buryatense 5GB1. Based on the homologous recombination and high transformation efficiency, gene deletion and heterologous gene expression can be simultaneously achieved by direct electroporation of PCR-generated linear DNA fragments. In this study, the influence of several key parameters (competent cell preparation, electroporation condition, recovery time, and antibiotic concentration) on the transformation efficiency was investigated for optimum conditions. The maximum electroporation efficiency of 719 ± 22.5 CFU/μg DNA was reached, which presents a 10-fold improvement. By employing this method, an engineered M. buryatense 5GB1 was constructed to biosynthesize isobutyraldehyde by replacing an endogenous fadE gene in the genome with a heterologous kivd gene. This study provides a potential and efficient strategy and method to facilitate the cell factory construction of methanotrophs.

Preparation of electrocompetent cells v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Transformation Efficiency ◽  
High Transformation Efficiency ◽  
High Transformation

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

Preparation of electrocompetent cells v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Transformation Efficiency ◽  
High Transformation Efficiency ◽  
High Transformation

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

Preparation of electrocompetent cells v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Transformation Efficiency ◽  
High Transformation Efficiency ◽  
High Transformation

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

scholarly journals Development of strategy for competent cell preparation and high efficiency plasmid transformation using different methods

2011 ◽  
Vol 29 (1) ◽  
pp. 17 ◽  
Author(s):  
Anubhuti Sharma ◽  
Arushi Girdhar ◽  
Nidhi Srivastava
Keyword(s):  
High Efficiency ◽  
Transformation Efficiency ◽  
Great Influence ◽  
Competent Cell ◽  
Cell Preparation ◽  
Site Specific ◽  
E Coli ◽  
Cost Efficient ◽  
Normal Laboratory ◽  
Complete Genomic

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible. With the recent availability of complete genomic sequences of many organisms and plants, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. This study compares two different methods of preparation of competent cells using two strains of E. coli DH5α and HB101. From results the most efficient strain was found DH5α for cloning as it supports blue white screening utilizing galactosidase activity. The concentration of calcium chloride is another important factor; various concentrations of CaCl2 were tried. Optimum concentration was found to be 75 mM. However PEG also has great influence on transformation efficiency, use of 40% PEG gave the best transformation efficiency. A convenient and rapid method for the genetic transformation of E. coli with appropriate plasmid is proposed which can be utilised for high efficiency transformation in normal laboratory conditions.

scholarly journals Electroporation-Based Genetic Manipulation in Type I Methanotrophs

2016 ◽  
Vol 82 (7) ◽  
pp. 2062-2069 ◽  
Author(s):  
Xin Yan ◽  
Frances Chu ◽  
Aaron W. Puri ◽  
Yanfen Fu ◽  
Mary E. Lidstrom
Keyword(s):  
Process Development ◽  
Transformation Efficiency ◽  
Genetic Manipulation ◽  
Industrial Process ◽  
Type I ◽  
Content Type ◽  
High Transformation Efficiency ◽  
New Antibiotics ◽  
Industrial Strains ◽  
High Transformation

ABSTRACTMethane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs:Methylomicrobium buryatense5GB1C,Methylomonassp. strain LW13, andMethylobactertundripaludum21/22. We further developed this technique inM. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency ofM. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) andsacBcounterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation ofM. buryatenseand other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.

scholarly journals A method for the large scale isolation of high transformation efficiency fungal genomic DNA

1996 ◽  
Vol 145 (2) ◽  
pp. 261-265 ◽  
Author(s):  
Donglu Zhang ◽  
Yifan Yang ◽  
Lisa A. Castlebury ◽  
Carl E. Cerniglia
Keyword(s):  
Genomic Dna ◽  
Large Scale ◽  
Transformation Efficiency ◽  
High Transformation Efficiency ◽  
High Transformation
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