High transformation efficiency of Bacillus subtilis with integrative DNA using glycine betaine as osmoprotectant

2012 ◽  
Vol 424 (2) ◽  
pp. 127-129 ◽  
Author(s):  
Fatma Meddeb-Mouelhi ◽  
Carlos Dulcey ◽  
Marc Beauregard
Keyword(s):  
Bacillus Subtilis ◽  
Glycine Betaine ◽  
Transformation Efficiency ◽  
High Transformation Efficiency ◽  
High Transformation

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 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.

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

scholarly journals High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 356 ◽  
Author(s):  
Yiran Wang ◽  
Hoda Yaghmaiean ◽  
Yuelin Zhang
Keyword(s):  
Target Genes ◽  
Bacterial Culture ◽  
Transformation Efficiency ◽  
Transformation Rate ◽  
High Transformation Efficiency ◽  
Floral Dip ◽  
Large Numbers ◽  
High Transformation ◽  
Transformation Methods ◽  
Inoculation Medium

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD600≈2.0) in large cultures and resuspended in inoculation medium to OD600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees relatively high transformation rate in Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

Sign in / Sign up

Export Citation Format

Share Document