The authors are grateful to their colleagues and staff of present and past laboratories that have supported the development of this technique. S.P.'s laboratory is supported by the Canadian Institute for Health Research Operating Grant MOP-84473 and Alberta Innovates-Health Solutions (funded by the Alberta Heritage Foundation for Medical Research Endowment Fund). D.P.'s was supported by National Institutes of Health grants MD001091-01 and GM068855-02.

1. Preparation of Bacterial Cultures, Tools, and Reagents (DAY 1, Afternoon)
<ol>
	<li>In the late afternoon, inoculate 1-5 ml autoclaved LB broth in sterile (for example, 100 x 13 mm) borosilicate glass test tubes with a small aliquot of bacteria (E. coli or V. cholerae).</li>
	<li>Set the inoculated test tubes in a roller drum housed at 37 &deg;C temperature (warm room or incubator), turn on the roller drum at high speed, and incubate O/N.</li>
	<li>Prepare a cell spreader in the shape of a "hockey stick" from a glass rod by heating the middle of the rod in the flame of a Bunsen burner until the glass softens and then direct the bend gently with tweezers or pliers into a 135&deg; angle. Heat the rod once more in the middle point between the end and the first bend and bend it at a 45&deg; angle inwards (in the opposite direction of the first bend).</li>
	<li>Prepare LB-agar without antibiotics and pour into Petri dishes in preparation of the electrocompetence protocol, and LB-agar plates containing the appropriate antibiotic (according to the resistance marker on the vector to be electroporated), and store at 4 &deg;C.</li>
	<li>Prepare a filter sterilized 2 mM CaCl2 solution for V. cholerae, or autoclave ddH2O for E. coli, and store at 4 &deg;C.</li>
</ol>
2. Growth of Electrocompetent Bacteria (DAY 2, Morning)
<ol>
	<li>Deliver 100 &mu;l of the O/N bacterial culture onto each LB agar plate, frozen glycerol stocks may also be utilized and directly delivered onto the plate.</li>
	<li>Dip the hockey stick-shaped cell spreader in 100% EtOH, briefly drain and burn off the remaining EtOH to sterilize it prior to use and spread. Spread the 100 &mu;l O/N bacterial culture evenly with the sterile glass cell spreader making sure not to disrupt (break) the agar surface.</li>
	<li>Incubate the plate at 37 &deg;C for 4-6 hr, or until a thin lawn of bacterial growth becomes distinguishable. Cells are most competent when actively growing.</li>
</ol>
3. Preparation of Electrocompetent Bacterial Cells (DAY 2, Afternoon)
<ol>
	<li>Harvest the bacteria with a sterile inoculating loop making sure not to pierce or break the surface of the agar. One 2 mm diameter bacterial mass is sufficient for a single transformation. Typically, this requires scraping the surface of the thin lawn with the inoculating loop two or three times. Several samples (typically between 4-6) may be collected from the same plate.</li>
	<li>Resuspend the bacterial mass in 1 ml ice-cold 2 mM CaCl2 (for V. cholerae) or sterile ddH2O (for E. coli) and mix well until no clumps are visible, keep on ice.</li>
	<li>Centrifuge each bacterial suspension for 5 min at 5,000 x g in a refrigerated microcentrifuge set to 4 &deg;C, or in a microcentrifuge stored in a cold room set to 4 &deg;C.</li>
	<li>Discard the supernatant, resuspend the bacterial pellet in the same volume of ice-cold 2 mM CaCl2 or sterile ddH2O, and repeat the centrifugation step as done before twice more for a total of three washes.</li>
	<li>Remove supernatant, resuspend, and then loosen the bacterial pellet thoroughly in 40 &mu;l ice cold 2 mM CaCl2 or sterile ddH2O and keep on ice.</li>
</ol>
4. Transformation of Electrocompetent Bacteria (DAY 2, Afternoon)
<ol>
	<li>Add up to 1 &mu;g of plasmid DNA (in up to 1 &mu;l water or Tris-EDTA buffer) to the 40 &mu;l bacterial suspension and transfer this mixture into a pre-chilled, sterile 0.2 cm gap cuvette. The salt concentration in the DNA sample must be lowa, as it will contribute to arcing of the pulse in the next step.</li>
	<li>Insert the cuvette into the electroporation chamber of the pulse control module, and electroporate at 1.8 kV, 25 &mu;F. The time constant should be ~5.0 msec, and no arcing should occur.</li>
	<li>Quickly recover the cell suspension by resuspending into 1 ml LB broth and transfer into previously autoclaved borosilicate glass test tube.</li>
	<li>Allow the cells to recover by incubating under aerated growth conditions (in a roller drum) at 37 &deg;C for 30 min without antibiotic selection.</li>
	<li>Plate the bacteria onto the previously prepared LB agar plates in the presence of the appropriate selective agent (antibiotic), and incubate at 37 &deg;C O/N. If bacteria are transformed with a high concentration of purified vector (0.1-1 &mu;g supercoiled plasmid), deliver 10 &mu;l of bacterial culture onto the edge of the LB agar plate and with a sterile inoculating loop streak for isolated colonies using the quadrant streak method. If a ligation mixture was transformed, deliver 100 &mu;l of bacterial culture onto each agar plate and evenly spread it with the sterilized glass cell spreader.</li>
</ol>
a If the time constant is short (below 3.5 msec), or pulsing leads to arcing, then re-precipitate the DNA sample, wash with 70% EtOH twice to remove salts before drying and resuspending in TE buffer.

<ol>
	<li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1 (7), 841-845 (1982).</li><li>Dower, W. J., Miller, J. F., Ragsdale, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+E.+coli+by+high+voltage+electroporation.">High efficiency transformation of E. coli by high voltage electroporation.</a> Nucleic Acids Res. 16 (13), 6127-6145 (1988).</li><li>Taketo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transfection+of+Escherichia+coli+by+electroporation.">DNA transfection of Escherichia coli by electroporation.</a> Biochim. Biophys. Acta. 949 (3), 318-324 (1988).</li><li>Fiedler, S., Wirth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+bacteria+with+plasmid+DNA+by+electroporation.">Transformation of bacteria with plasmid DNA by electroporation.</a> Anal. Biochem. 170 (1), 38-44 (1988).</li><li>O'Callaghan, D., Charbit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Salmonella+typhimurium+and+Salmonella+typhi+by+electroporation.">High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation.</a> Mol. Gen. Genet. 223 (1), 156-158 (1990).</li><li>Hamashima, H., Nakano, T., Tamura, S., Arai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Vibrio+parahaemolyticus,+Vibrio+alginolyticus,+and+Vibrio+cholerae+non+O-1+with+plasmid+DNA+by+electroporation.">Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae non O-1 with plasmid DNA by electroporation.</a> Microbiol. Immunol. 34 (8), 703-708 (1990).</li><li>Grones, J., Turna, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+microorganisms+with+the+plasmid+vector+with+the+replicon+from+pAC1+from+Acetobacter+pasteurianus.">Transformation of microorganisms with the plasmid vector with the replicon from pAC1 from Acetobacter pasteurianus.</a> Biochem. Biophys. Res. Commun. 206 (3), 942-947 (1995).</li><li>Allen, S. P., Blaschek, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-induced+transformation+of+intact+cells+of+Clostridium+perfringens.">Electroporation-induced transformation of intact cells of Clostridium perfringens.</a> Appl. Environ. Microbiol. 54, 2322-2324 (1988).</li><li>Belliveau, B. H., Trevors, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Bacillus+cereus+vegetative+cells+by+electroporation.">Transformation of Bacillus cereus vegetative cells by electroporation.</a> Appl. Environ. Microbiol. 55, 1649-1652 (1989).</li><li>Chassy, B. M., Flickinger, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Lactobacillus+casei+by+electroporation.">Transformation of Lactobacillus casei by electroporation.</a> FEMS Microbiol. Lett. 44, 173-177 (1987).</li><li>Dunny, G. M., Lee, L. N., LeBlanc, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+electroporation+and+cloning+vector+system+for+gram-positive+bacteria.">Improved electroporation and cloning vector system for gram-positive bacteria.</a> Appl. Environ. Microbiol. 57 (4), 1194-1201 (1991).</li><li>Micheletti, P. A., Sment, K. A., Konisky, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+coenzyme+M-auxotrophic+mutant+and+transformation+by+electroporation+in+Methanococcus+voltae.">Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus voltae.</a> J. Bacteriol. 173 (11), 3414-3418 (1991).</li><li>Schleper, C., Kubo, K., Zillig, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+particle+SSV1+from+the+extremely+thermophilic+archaeon+Sulfolobus+is+a+virus:+demonstration+of+infectivity+and+of+transfection+with+viral+DNA.">The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA.</a> Proc. Natl. Acad. Sci. U.S.A. 89 (16), 7645-7649 (1992).</li><li>Aune, T. E., Aachmann, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+to+increase+the+transformation+efficiencies+and+the+range+of+bacteria+that+can+be+transformed.">Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed.</a> Appl. Microbiol. Biotechnol. 85 (5), 1301-1313 (2010).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transformation+of+Escherichia+coli+with+plasmids.">Studies on transformation of Escherichia coli with plasmids.</a> J. Mol. Biol. 166, 557 (1983).</li><li>Unterweger, D., Kitaoka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+Type+VI+Secretion+System+Expression+Gives+Vibrio+cholerae+Intra-+and+Interspecific+Competitive+Advantages.">Constitutive Type VI Secretion System Expression Gives Vibrio cholerae Intra- and Interspecific Competitive Advantages.</a> PLoS One. 7 (10), e48320 (2012).</li></ol>

No disclosures are applicable.

Transformation efficiency is subject to a variety of factors regardless of the method employed to prepare competent cells. Analogous variables apply to this method; great care must be taken that once the cells are harvested from the agar plate such that they are maintained at constant cold temperature (no higher than 4 &deg;C). The bacterial lawn must be actively growing at the time of collection; cells must be in mid-logarithmic phase of growth. Quality of DNA (i.e. contamination of salts) influences transformation efficiency. Specifically, when employing this method, the agar should not be broken as agar fragments in the cuvette will cause arcing of the pulse.
Most problems encountered with this technique are associated with two issues; collecting the bacteria during the proper window of time from the agar plate when they are actively growing. Collecting them too soon will yield insufficient cell numbers and will render the scraping off the LB agar plate frustrating because the "pellicule" they form is hard to lift off the surface of the plate. Collecting bacteria too late will decrease their competence dramatically. The window of time will naturally vary depending on the density of the inoculum plated on the LB agar plates. The second critical step is to keep the bacteria cold (4 &deg;C) from the moment they are lifted off the agar plate and resuspended in CaCl2 or ddH2O until they are plated on the LB agar following transformation. The microfuge tubes containing the bacteria, cuvettes and buffer should be pre-chilled and kept on ice at all times until the bacteria are plated. However, additional problems can arise when the sterile ddH2O or CaCl2 contain precipitate or are contaminated. For this reason we recommend to prepare this reagent fresh.
Troubleshooting transformation is facilitated by including controls when transforming bacteria rendered competent by any method. A negative control consisting of a batch of non-transformed competent bacteria plated on an LB agar plate containing the selective marker will rapidly help determine whether the antibiotic on the plate is effective. A positive control transformation with a known plasmid preparation of good quality and quantity harboring the same selective marker as the experimental sample will be helpful in determining whether the bacteria are competent but also whether the experimental DNA transformed was sufficient in quantity or quality.
The advantages of this technique are multiple; the method can be carried out within the same day that a transformation is planned for. We have found that frozen bacterial glycerol stock may be plated directly on the LB agar without any loss in electrocompetence; we have done this in "emergency" situations (after forgetting to start the overnight culture the day before). Although we could not test every strain known, any E. coli or V. cholerae strain that can be transformed by electroporation has been amenable to this protocol in our hands. The technique is rapid, effective, and yields transformation efficiencies comparable to those obtained from preparing electrocompetent bacteria employing traditional methods. The method eliminates the requirement for high speed centrifuge and associated rotors, an orbital incubator shaker and sterile centrifuge buckets (containers), and large volumes of autoclaved buffer. Although the Table of Specific Reagents and Equipment is quite comprehensive, most laboratories that carry out electroporation will have most, if not all, of these materials already available. Perhaps most importantly, the technique can be readily applied to generate electrocompetent laboratory-specific strains with mutant backgrounds rapidly. We are unaware of any limitations of this technique as it has replaced the traditional method of preparation of electrocompetent bacteria in our laboratories.
The results of our experiments shown here were carried out with fresh electrocompetent cells produced with the traditional method that had been stored at -80 &deg;C for no longer than 1 week. However, frozen electrocompetent cell stocks lose competence and or viability over extended periods of storage. Our abridged protocol permits the preparation of fresh electrocompetent cells the same day the transformation is planned without concerns about age of the stock which may result in decreased transformant yield. We have not attempted storing electrocompetent cells produced using the rapid method for use on another day as this has not been necessary.
While not shown here, the efficiency of the electrocompetent cells prepared using our rapid method is sufficient for ligation mixtures. The transformations reported for non-O1 V. cholerae in a recent article by Unterweger et al.17 were carried out using electrocompetent bacteria prepared by the method described here (except those carried out by conjugation). This technique affords researchers to transform strains with mutant backgrounds rapidly and effectively without having to prepare large batches of cells. This rapid preparation of electrocompetent bacteria method might be adapted to other prokaryotes that have been shown to be amenable to electroporation as the principle of preparation and the electroporation settings are going to be the same as those of traditional protocols.

Since its' inception in the early 1980s1, electroporation has become an integral molecular biology technique, likely the single most common method employed to transform/transfect live cells with circular or linear nucleic acids. Originally developed for transfection of eukaryotic cells1, electroporation was subsequently adapted for transformation of E. coli2, 3. In bacteriology, electroporation has become the laboratory standard and has been modified to transform a wide range of bacteria including Gram-negative Pseudomonas4, Salmonellae5, Vibrios6, Serratiae, and Shigellae7; Gram-positive Clostridia8, Bacilli9, Lactobacilli10 and Enterococci11. Even some Archaebacteria have proven amenable to transformation by electroporation, including Methanococci12 and Sulfolobus species13. For a comprehensive review please refer to Aune and Aachmann14. Efficiency of transformation by electroporation is reportedly 10-20 fold higher than chemical competence or heat shock2. However, much like preparing bacteria for chemical competence as optimized by Douglas Hanahan in the 1980s15, cells must also be prepared to be electrocompetent.
Electroporation is a rapid and effective means of bacterial transformation, requiring electrocompetent bacteria, purified DNA, a pulse control module that delivers the electrical impulse, and a chamber that accommodates small disposable cuvettes that function as an electrode. Briefly, cold, competent bacteria are mixed with plasmid DNA, subjected to a high-voltage pulse in a cuvette, resuspended in growth media, incubated at 30-37 &deg;C for 30-45 min, and then plated on semisolid medium (nutrient agar plates) with the appropriate selective antibiotic.
In contrast to the pulsing step, the preparation of electrocompetent E. coli remains a multipart procedure that is traditionally carried out in large volumes. Briefly, an overnight culture of bacteria is inoculated into an Erlenmeyer flask with 100 ml to 1 L Luria Broth (LB), grown to mid-logarithmic phase of growth (OD600 of &lt;1.0), and subjected to serial washes with a sterile non-ionic buffer or double-distilled water (ddH2O) in decreasing volumes at 4 &deg;C. Great care must be taken to keep the cells cold throughout the entire procedure and prevent contamination. This requires the use of autoclaved centrifugation buckets and non-ionic buffers or ddH2O, an orbital shaker incubator, a large volume high-speed refrigerated centrifuge and a set of rotors. Bacteria are finally resuspended in a small volume of buffer supplemented with 10% glycerol and aliquoted into ~40 &mu;l volumes, which are stored at -80 &deg;C until use. Low-temperature storage often results in decreased transformation efficiencies due to loss of viability over time.
Electrocompetent bacterial cells are also available from a variety of commercial sources but only for a limited number of (often recombination-deficient) E. coli strains commonly employed as hosts to propagate a wide range of plasmids. As a result researchers rely on in-house methods to prepare their own strains/mutants for transformation.
An alternate, rapid and efficient protocol for the preparation of electrocompetent E. coli has been in use by a few molecular bacteriology laboratories for some time now and has been extended to additional Gram-negative bacteria, in our case V. cholerae. The originator of the protocol cannot be identified as it has been optimized by other researchers over time. The method presented here has been used in our laboratories for nearly two decades, and it is our goal to share this useful protocol with our peers.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the Equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Gene Pulser Module</td>
			<td>Bio-Rad</td>
			<td>165-2668</td>
		</tr>
		<tr>
			<td>Gene Pulser Cuvette Chamber</td>
			<td>Bio-Rad</td>
			<td>165-2669</td>
		</tr>
		<tr>
			<td>Electrocuvettes (0.2 cm gap)</td>
			<td>Bio-Rad</td>
			<td>165-2082</td>
		</tr>
		<tr>
			<td>13 x 100 mm Pyrex Rimless Culture Tubes</td>
			<td>Corning</td>
			<td>70820-13</td>
		</tr>
		<tr>
			<td>13 mm Culture Tube Closures</td>
			<td>Cole Parmer</td>
			<td>EW-04500-00</td>
		</tr>
		<tr>
			<td>6 x 250 mm Glass Rod</td>
			<td>Lab Source</td>
			<td>11381D</td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-12</td>
		</tr>
		<tr>
			<td>Roller Drum Motor Assembly</td>
			<td>New Brunswick Scientific</td>
			<td>M1053-4004</td>
		</tr>
		<tr>
			<td>Roller Drum for 13 x 100 mm Culture Tubes</td>
			<td>New Brunswick Scientific</td>
			<td>M1053-0306</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Chemicals</td>
			<td>C5670</td>
		</tr>
		<tr>
			<td>Ethanol, anhydrous/denatured</td>
			<td>Sigma Chemicals</td>
			<td>227649</td>
		</tr>
		<tr>
			<td>Inoculating Loop</td>
			<td>Decon Labs</td>
			<td>MP182-5</td>
		</tr>
		<tr>
			<td>Refrigerated Microcentrifuge 5451 R</td>
			<td>Eppendorf</td>
			<td>22621425</td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Fisher Scientific</td>
			<td>03-917Q</td>
		</tr>
		<tr>
			<td>LB Broth MILLER</td>
			<td>EMD</td>
			<td>1.10285.0500</td>
		</tr>
		<tr>
			<td>Bacteriological Agar</td>
			<td>Fisher Scientific</td>
			<td>S25127</td>
		</tr>
	</tbody>
</table>

rapid protocol for preparation of electrocompetent escherichia coli and vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

We carried out a set of transformations with E. coli and V. cholerae to compare transformation efficiency of our rapid methodology with an adaptation of the (traditional) method originally described by Dower et al.2 for the preparation of electrocompetent bacteria. To carry out the traditional preparation of competent cells, 500 ml LB in a 2 L-Erlenmeyer flask were inoculated with 500 &mu;l overnight culture of E. coli DH5&alpha; or V. cholerae O395, incubated in an orbital shaker (215 rpm at 37 &deg;C) and harvested at OD600 of between 0.5-1.0. The flask was cooled on ice and the cultures centrifuged in a pre-chilled rotor at 4,000 x g for 10 min at 4 &deg;C. The cell pellets were thoroughly resuspended in 500 ml ice-cold 2 mM CaCl2 for V. cholerae and ddH2O for E. coli, and centrifuged again under the same conditions. Subsequent rounds of resuspension and centrifugation were carried out in decreasing volumes of 200 and 100 ml volumes making sure the culture remained chilled at 4 &deg;C. Finally, a resuspension and centrifugation in a 10 ml volume was carried out with 10% glycerol. The final pellet was resuspended in 2 ml 10% glycerol and 40 &mu;l aliquots were frozen at -80 &deg;C and stored no longer than one week prior to electroporation.
Electroporation conditions including pulse settings and cuvette size were identical for both bacterial species in all electroporations regardless of method of preparation of competent cells. The only difference was that we carried out the washes in cold ddH2O for E. coli and cold 2 mM CaCl2 for V. cholerae. Table 1 shows results of bacteria transformed by electroporation identically but rendered competent either with the rapid protocol or with the traditional method. We employed strain O395 and DH5&alpha; as commonly used, representative strains of V. cholerae and E. coli respectively; similar results can be obtained with other strains of the same species (albeit not every single strain has been tested) and likely adapted to other Gram-negative bacteria and probably beyond.
To insure that equal cell numbers were present in each batch pulsed, electrocompetent bacteria generated utilizing the rapid method were pooled upon collection, mixed, kept homogenously suspended, and aliquoted in 40 &mu;l volumes shortly prior to electroporation. Electrocompetent bacteria generated utilizing the traditional method were treated similarly prior to freezing in 40 &mu;l aliquots. Following delivery of the pulse, each batch of 40 &mu;l electroporated bacteria was suspended into 400 &mu;l LB and serially diluted 1:10. One hundred microliters of each dilution were plated on LB agar in the presence of 100 &mu;g/ml ampicillin and spread using a sterile glass cell spreader and incubated at 37 &deg;C overnight. To quantify the total number of bacteria employed in each transformation, 40 &mu;l volumes of electrocompetent bacteria of each prepared batch were serially diluted, and plated on LB agar without antibiotics identically in parallel. One aliquot of equally treated bacteria from each batch was electroporated with 1 &mu;l Tris-EDTA (TE) buffer but without DNA (mock transformation), serially diluted and plated on LB agar without antibiotics to estimate the number of cells lost by the delivery of the electric pulse. Lastly, one additional variation to the experiment was carried out to determine whether 30 min incubations at 37 &deg;C in 1 ml LB after electroporation but prior to plating on LB-agar would affect the recovery of transformed cells. colony forming units (CFUs) were enumerated on the following day.
For this experiment we employed pUC18, a common laboratory plasmid, and batches of competent bacteria consisting of approximately 108 CFUs for cells prepared utilizing the traditional method and 107 CFUs for cells prepared utilizing the rapid protocol. Electroporation alone (without DNA) reduced viable CFUs by 10-100 fold irrespective of the method used to prepare the electrocompetent cells. As shown in Table 1, transformant yield was within the 106 -107 CFUs/&mu;g of DNA range in three replicate experiments (standard deviations in parentheses) for E. coli (DH5&alpha;) and V. cholerae (O395) respectively using the traditional, lengthier method for electrocompetent cell preparation. Transformant yield appeared decreased 10 fold to 105 -106 CFUs /&mu;g of DNA in three replicate experiments (standard deviation in parentheses) for E. coli and V. cholerae respectively using the rapid method. For this reason we determined the percentage of transformed bacteria by dividing transformed CFUs by the number of CFUs recovered from "mock transformations" (without plasmid) from otherwise identical batches of competent cells. The percentage of transformed bacteria was within one log (2.5-9.4%) regardless of method of preparation and species transformed (Table 1 bracketed numbers). These results suggest that the efficiency of the rapid method is comparable to that of the traditional lengthier procedure of preparing competent cells and that the representative strains of Vibrio cholerae and Escherichia coli are equally amenable to the rapid procedure.
We found that transformation efficiency, for plasmids like pUC18 harboring &beta;-lactamase cassettes, is not affected by the 30 min recovery time in LB broth. The same number of transformants was recovered whether the cells were plated to LB agar in the presence of ampicillin directly, after electroporation, or whether they were allowed a 30-45 min recovery time in LB broth at 37 &deg;C prior to plating. Our findings that ampicillin-resistance does not require outgrowth under nonselective conditions suggests that &beta;-lactamase expression occurs sufficiently fast upon transformation. However, it should be noted that we carried out serial dilutions of pulsed bacteria in LB broth, which may have contributed to recovery from the electric shock and initiate gene expression.
<img alt="Figure 1" src="/files/ftp_upload/50684/50684fig1.jpg" /> 
Figure 1. Graphical depiction of the methodology for the rapid preparation of electrocompetent bacteria. After 4-6 hr (depending on the density of the inocula) sufficient numbers of bacteria can be collected to carry out four to six transformations from a single LB agar plate. To insure equal number of competent cells for each final volume to be electroporated, bacteria collected from the LB agar plate were initially pooled and washed together then aliquoted in 40 &mu;l volumes depending on the size of the pellet (larger pellets were diluted in larger volumes divisible by 40 &mu;l).
<table border="1">
	<tbody>
		<tr>
			<td colspan="3">Number of Ampicillin-resistant transformants/&mu;g DNA (Ampicillin-resistant transformants recovered [%])</td>
		</tr>
		<tr>
			<td>Strain</td>
			<td>Traditional method</td>
			<td>Rapid method</td>
		</tr>
		<tr>
			<td>E. coli (DH5&alpha;)</td>
			<td>2.3 x 106 (&plusmn; 7.3 x 105) [9.4%]</td>
			<td>3 x 106 (&plusmn; 7.1 x 105) [2.5%]</td>
		</tr>
		<tr>
			<td>V. cholerae (O395)</td>
			<td>4 x 107 (&plusmn; 1.7 x 107) [5%]</td>
			<td>6.7 x 105 (&plusmn; 2.1 x 105) [3.3%]</td>
		</tr>
	</tbody>
</table>
Table 1. Transformation efficiency per microgram of DNA (pUC18) of electrocompetent bacteria prepared by the traditional method compared to the rapid method.
All transformations were carried out with 500 ng pUC18 DNA (~80% supercoiled DNA as visualized by 1% agarose gel electrophoresis) and the electroporated cells were serially diluted 10-fold in LB broth prior to plating for CFU on LB-agar with antibiotics. Controls of non-electroporated bacteria and of bacteria electroporated without plasmid, serially diluted and plated on LB agar without antibiotics were carried out for each set of transformations to determine the total number of viable bacteria prior and after pulsing. The electroporated controls represented the base line for the percent of viable bacteria successfully transformed.

Watch this Scientific Journal Video about Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae at JoVE.com

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

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76.2K Views.  University of Texas Brownsville. Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Video: Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...