Easy Transformation of Escherichia coli: Nanoparticle-Mediated Transformation
Abstract
In this protocol, a colloidal solution containing mineral nanofibers is mixed with Escherichia coli and plasmid DNA and plated immediately on the appropriate selective plates. Sliding frictional forces, created when the bacteria are spread with a polystyrene stir stick across the surface of the agar, may result in penetration of the bacteria by the mineral fibers with its adherent cargo of DNA. The number of transformants increases during the first 60 sec of exposure to the mineral fiber:DNA complex and then reaches a plateau of ∼1 × 106 to 2 × 106 transformants/milligrams of DNA. Although not highly efficient, the method is very simple.
MATERIALS
Reagents
E. coli culture (e.g., TOP10 cells or another robust strain) in log phase
HEPES (1 m, pH 7.4)
KCl (1 m)
Plasmid DNA
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A standard preparation of a plasmid DNA should be stored in aliquots and used to measure the competence of batches of chemically treated E. coli. pUC19, which is commonly used for this purpose, is available from New England Biolabs.
Sepiolite
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Spanish sepiolite with a mean fiber length of 2 mm and a mean diameter of 20 nm is available from Kremer Pigmente GmbH & Co, KG, Germany (catalog no. 58945)
SOB, SOC, or LB agar plates (1%–2%) containing the appropriate antibiotic
Equipment
Incubator (37°C)
Polystyrene stick (available from Sarstedt, Germany)
METHOD
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See Box 1 for descriptions of essential controls.
ESSENTIAL CONTROLS FOR BACTERIAL TRANSFORMATION
In every experiment, it is essential to include positive controls to measure the efficiency of transformation, and negative controls to eliminate the possibility of contamination and to identify the potential causes of failure.
Negative Controls
An aliquot of competent cells to which no DNA is added should be carried through the transformation experiment. The entire aliquot should be plated on a single agar plate containing the appropriate antibiotic used to select transformants. No bacterial colonies should grow on this plate or on a selective plate that received no bacteria at all. If any are detected, the following possibilities should be considered.
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The competent cells are contaminated with an antibiotic-resistant strain of bacteria during the experiment. Perhaps one of the solutions/reagents used in the transformation protocol is contaminated.
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The selective plates are defective. Perhaps the antibiotic was omitted altogether from the plates or was added to agar that was too hot.
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The selective plates are contaminated with an antibiotic-resistant strain of bacteria. In this case, colonies usually appear both on the surface of the medium and in the agar.
Positive Controls
An aliquot of competent cells should be transformed with a known amount of a standard preparation of circular superhelical plasmid DNA. This control provides a measure of the efficiency of transformation and allows a standard for comparison with previous transformation experiments.
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1. Autoclave a suspension of 0.01% sepiolite in 200 mm KCl and 5 mm HEPES (pH 7.4).
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2. Briefly centrifuge 500 mL of a log-phase culture of E. coli.
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3. Resuspend the bacterial pellet in 100 mL of sepiolite suspension, and add 50 ng of plasmid DNA. Wait for 60 sec and then use a polystyrene stick to spread the resulting suspension on a 1%–2% agar plate (at least 2 d old) containing the appropriate antibiotic. Spreading should be continued for ∼30 sec after the liquid has soaked into the agar, when the frictional resistance to spreading increases.
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4. Incubate the plate overnight at 37°C.
DISCUSSION
Yoshida's group in Miyazaki published a series of papers confirming and extending the observation by Appel et al. (1988) that transformation of E. coli by plasmids can be accomplished using mineral nanofibers (Yoshida et al. 2001, 2002, 2007; Yoshida and Sato 2009 [and references therein]). Previously, an impediment to the wider adoption of this method was the nature of the mineral fibers used as the transfection agents: Both Appel et al. (1988) and Yoshida's group used chrysotile, a form of asbestos. More recently, however, Yoshida's observations were confirmed (Wilharm et al. 2010) using a suspension of noncarcinogenic fibers (Spanish sepiolite) instead of the potentially dangerous chrysotile whisker used by Yoshida and his coworkers. The basic protocol outlined by Wilharm et al. (2010) is above.
Footnotes
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From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.