Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens

Abstract

Electroporation entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous macromolecules, which may otherwise be excluded. Removal of the external field leads to the resealing of the membrane electropores permitting the survival of the electrically stimulated recipient cells. Using this technique foreign deoxyribonucleic acid (DNA) has been successfully introduced into many cell types both from prokaryotes and eukaryotes. Increase in pulse voltage and length beyond a critical limit has been reported to decrease transformation efficiency, hence in this study we have investigated another strategy i.e. increase in the number of pulses at constant high voltage and pulse duration. Commonly used Agrobacterium strains LBA4404 and EHA101 and binary vector pCAMBIA1301 were used. Transformants were selected on a combination of hygromycin and kanamycin, and confirmed by polymerase chain reaction (PCR) and restriction analysis. Increase in the number of pulses was found to show a significant and linear increase in transformation efficiency.Application of deoxyribonucleic acid (DNA) technology to genetically manipulate cellular functions requires a suitable high efficiency transformation system. There are many approaches available for introducing foreign DNA into eukaryotic and prokaryotic cells, however electroporation is the most commonly used technique and currently of more practical interest. It is a gene transfection technique, which entails brief, high intensity pulse to facilitate the entry of exogenous molecules like DNA, ribonucleic acid (RNA), and protein. Removal of the external field leads to the resealing of the membrane electropores.Applying high intensity electric fields to reversibly permeablize biomembranes to create structural distortions, allowing the uptake of DNA was first demonstrated by Neuman in mouse myeloma cells (Neuman et al. 1982).

Construction of an Engineering Strain Producing High Yields of α-TransglucosidaseviaAgrobacterium tumefaciens-Mediated Transformation ofAsperillus niger

“…21) Initially, binary vector pBI-hph, having the hph gene as selection marker, was introduced into A. tumefaciens LBA4404 by electroporation. 30) The transformants were isolated on YEB agar plates supplemented with Rif (50 mg/mL), Str (20 mg/mL), and Kan (50 mg/mL), and confirmed by PCR and enzyme digestion. Positive A. tumefaciens transformants were cultured in 5 mL of YEB on a rotatory shaker (180 r/min) at 28 C for 20 h. A. tumefaciens cells were collected by centrifugation (5,000 r/min, 10 min) and resuspended in fresh YEB containing AS (200 mmol/L) at a density of OD 600 ¼ 0:15: After preincubation for 6-10 h at 28 C on a shaker to an OD 600 of 0.4 to 1.0, 100 mL of the cell suspension was mixed with an equal volume of spore suspension at a concentration of 10 5 spores per milliliter to 10 8 spores per milliliter, and then the mixture was spread onto filter paper placed on IM agar plate (with 200 mmol/L AS).…”

“…Binary vector pBI-Glu, including the hph gene and the -transglucosidase expression cassette, was transferred into A. tumefaciens LBA4404 via electroporation, 30) and the positive transformants were used to infect A. niger TCCC41056 by ATMT, which was established and optimized in the above experiments for random insertional mutagenesis.…”

Insecticide susceptibility status in three medically important species of mosquitoes, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus, from Bruhat Bengaluru Mahanagara Palike, Karnataka, India

“…Recombinant binary vector (pKM12GW) carrying three gene cassettes (CaMV35S::GUS-Tnos, CaMV35S::cry1Aabc-Tnos and CaMV35S::cry1Ec-Tnos) was used for transformation of Agrobacterium tumefaciens (strain EHA105) by electroporation [22]. For electroporation, the binary vector (200 ng) was mixed with 100 ll (OD 590 0.66) competent cells and exposed to electric pulse of 1,440 V cm -1 , duration of *5 ms (Eppendorf, Hamburg, Germany) and pulse was repeated after 15-20 s twice.…”

Isolation and expression analysis of a LEAFY/FLORICAULA homolog and its promoter from London plane (Platanus acerifolia Willd.)

“…The constructed vector was verified by digestion with SpeI and SalI, and by PCR amplification using primers LFYF and 1303R (Table 1). Subsequently, the pPlacL-FY::GUS construct was introduced into the Agrobacterium tumefaciens strain EHA105 by an electroporation method (Mahmood et al 2008). …”