Preparation of electrocompetent cells v2

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

Preparation of electrocompetent cells v2

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

Preparation of electrocompetent cells v2

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

Preparation of electrocompetent cells v1

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

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

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.

High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

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 (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=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.

High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

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.

Free fatty acids promote transformation efficiency of yeast

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.

A novel method for genetic transformation of C. albicans using modified-hydroxyapatite nanoparticles as a plasmid DNA vehicle

A new method for delivery of plasmid DNA into Candida albicans using arginine–glucose–PEG functionalized hydroxyapatite nanoparticles as the vehicle which delivers pDNA with high transformation efficiency.

Agrotransformation of Phytophthora nicotianae: a simplified and optimized method

ABSTRACT Phytophthora nicotianae is a plant pathogen responsible for damaging crops and natural ecosystems worldwide. P. nicotianae is correlated with the diseases: citrus gummosis and citrus root rot, and the management of these diseases relies mainly on the certification of seedlings and eradication of infected trees. However, little is known about the infection strategies of P. nicotianae interacting with citrus plants, which rises up the need for examining its virulence at molecular levels. Here we show an optimized method to genetically manipulate P. nicotianae mycelium. We have transformed P. nicotianae with the expression cassette of fluorescence protein DsRed. The optimized AMT method generated relatively high transformation efficiency. It also shows advantages over the other methods since it is the simplest one, it does not require protoplasts or spores as targets, it is less expensive and it does not require specific equipment. Transformation with DsRed did not impair the physiology, reproduction or virulence of the pathogen. The optimized AMT method presented here is useful for rapid, cost-effective and reliable transformation of P. nicotianae with any gene of interest.