Eporator from Eppendorf

February 21, 2013

Overall

Performance

Ease-of-Optimization

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Hospital Universitario Marqués de Valdecilla (HUMV) and Instituto de Formación e Investigación Marqués de Valdecilla (IFIMAV)
Microbiology
Principal Investigator
Optimization of electroporation parameters using the Eppendorf Eporator. (A) Relationship between numbers of transformants obtained per microgram of pDsRed DNA and per milliliter of S. liquefaciens competent cells, and voltage. Electrocompetent cells of S. liquefaciens were prepared using a standard procedure (10% ice-cold glycerol as washing buffer). Electroporations were performed in early growth stage cultures (optical density 0.5). Plasmid pC.IGdsRed that encodes the bacterially optimized fluorescent protein DsRed, was introduced by electroporation into a Serratia liquefaciens clinical strain. The electroporation was carried out at different voltages with a 5 ms pulse using the Eppendorf Eporator. To maintain the plasmid-encoded marker, S. liquefaciens was grown in the presence of ampicillin (100 ug/ml). The values represent means (number) ± standard deviations from three independent experiments. (B & C) Fluorescent bacteria. Micrographs were originally captured at low (×200) and high magnification (×1000). Bar indicates 2 um.

Company:

Eppendorf

Product Name:

Eporator®

Catalog Number:

4309000019

Image

Electrocompetent bacteria are required for electroporation, a process by which electrical pulses create pores that allow genetic material to permeate the bacterial membrane. In the field of cellular microbiology, we are always open to new ideas for projects aimed at understanding the interactions between bacterial pathogens and eukaryotic cells, and we often need fluorescent labeled bacteria. In some cases, we only need one transformant; in others, we need a high number of fluorescent colonies, and the number of bacterial species that have been used in our experiments continues to grow. We use Gram positive and Gram negative bacteria for the transformation with DNA constructs encoding fluorescent proteins. We prefer electroporation, which is quicker and simpler than other methods. We normally use GFP and DsRed plasmids with antibiotic resistance marker genes.

Experimental Design and Results Summary

Application

Generation of fluorescent bacteria via transformation with GFP and DsRed plasmids.

Starting Material

Electrocompetent bacteria

Protocol Overview

Using the Eporator? is very easy. A typical protocol is as below: (1) Prepare electrocompetent bacteria testing different growth conditions, washing buffers, time points (as described in ‘Tips’ below). (2) Set the electroporation conditions using the two arrow keys -Eporator?. (3) Aliquot 50 ?l of cells into microcentrifuge tubes. Optional, store unused aliquots immediately at -80°C. (4) Add 1 µl DNA to a prechilled cuvette (1 mm gap). (5) Add 40-50 µl electrocompetent cells. Homogenize by gently mixing with pipette several times. (6) Place the electroporation cuvette in the cuvette mount and insert into the Eporator?. (7) Press Start (a signal tone sounds after the discharge). (8) Remove the cuvette and immediately place 1ml of pre-heated medium into the cuvette and mix. (9) Transfer bacteria to a new tube and incubate for 1 hour with strong shaking. (10) Plate bacteria on solid medium containing the appropriate selective agent.

Tips

We always store our cuvettes at -20°C before electroporation. Empty tubes and tubes containing bacteria are always on ice. We pay special attention to the optimization of the following parameters to improve efficiency: a) Washing buffer. When setting up a new protocol, we routinely test different washing buffers and select the buffer that gives the best electroporation efficiency. b) Growth conditions of the bacteria (test time points during the bacterial growth phase in order to select those which render better efficiency). We have used bacteria from the following time points: from 3 to 5 hours post-inoculation at the exponential phase, or late time points (up to 10 hours) for some hard-to-transform bacterial strains (such as anaerobes or acidophilic bacteria).

Results Summary

High efficiency of transformation for both Gram negative and Gram positive bacteria. Most Enterobacteriaceae strains are relatively easy to transform by electroporation with the Eporator? by washing bacterial cells in 10% ice-cold glycerol and using pulses below 1900 V. For Gram positive bacteria, we can use also additives such as sucrose, sorbitol or glucose that may act as osmotic stabilizers. Transforming in the presence of these additives, however, requires more optimization and higher pulses, normally between 1800 and 2500 V.

Features Summary

Very compact and intuitive, with an integrated electroporation chamber that eliminates voltage arches and sparks (in comparison with other devices where the electroporation chamber is connected to the power source by a cable). Easy to use.

Additional Notes

We have tested different cuvette brands in this device with similar results. Clinical isolates and environmental bacteria were also easy transformed, but efficiency also depends on specific protocols.

Image Gallery

Optimization of electroporation parameters using the Eppendorf Eporator. (A) Relationship between numbers of transformants obtained per microgram of pDsRed DNA and per milliliter of S. liquefaciens competent cells, and voltage. Electrocompetent cells of S. liquefaciens were prepared using a standard procedure (10% ice-cold glycerol as washing buffer). Electroporations were performed in early growth stage cultures (optical density 0.5). Plasmid pC.IGdsRed that encodes the bacterially optimized fluorescent protein DsRed, was introduced by electroporation into a Serratia liquefaciens clinical strain. The electroporation was carried out at different voltages with a 5 ms pulse using the Eppendorf Eporator. To maintain the plasmid-encoded marker, S. liquefaciens was grown in the presence of ampicillin (100 ug/ml). The values represent means (number) ± standard deviations from three independent experiments. (B & C) Fluorescent bacteria. Micrographs were originally captured at low (×200) and high magnification (×1000). Bar indicates 2 um.

Summary

The Good

User-friendly and intuitive interface; USB port to export data; compact size; simplicity of design leads to less need for instruction and support

The Bad

None

The Bottom Line

A really well-designed and useful instrument if you prefer electroporation to other methods of transformation.

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