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

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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 ...

rapid-protocol-for-preparation-electrocompetent-escherichia-coli

Nanyang Technological University

Research

JoVE Journal

Biology

76.8K Views.  University of Texas Brownsville.  Elektroporatie is een algemeen gebruikte werkwijze voor het introduceren van DNA in bacteriën in een proces dat transformatie. Traditionele protocollen voor de bereiding van elektrocompetente cellen tijdrovend en arbeidsintensief. Dit artikel beschrijft een alternatieve, snelle en efficiënte werkwijze voor de bereiding van elektrocompetente cellen thans werkzaam sommige laboratoria. 

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

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Immunology and Infection

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Chemistry

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Assessment of Hsp70 Chaperone Activity Using Escherichia coli dnaK756 Cells

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

Biochemistry

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Measurement of Reporter Proteins in Limosilactobacillus reuteri DSM20016

Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for use as a chassis for synthetic biological endeavors within the gastrointestinal tract. The wide phenotypic and genotypic variation observed within the genus led to a recent reclassification and the introduction of 23 novel genera.
Due to the breadth of variations within the old genera, protocols demonstrated in one member may not work as advertised with other members. A lack of centralized information on how exactly to manipulate specific strains has led to a range of ad hoc approaches, often adapted from other bacterial families. This can complicate matters for researchers starting in the field, who may not know which information does or does not apply to their chosen strain.
In this paper, we aim to centralize a set of protocols with demonstrated success, specifically in&#160;the Limosilactobacillus reuteri strain designation F275 (other collection numbers: DSM20016, ATCC23272, CIP109823), along with troubleshooting advice and common issues one may encounter. These protocols should enable a researcher with little to no experience working with L. reuteri DSM20016 to transform a plasmid, confirm transformation, and measure system feedback in a plate reader via a reporter protein.

Author Spotlight: Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016  

Here, we present protocols for working with Limosilactobacillus reuteri DSM20016, detailing growth, plasmid transformation, colony PCR, fluorescent reporter protein measurement, and limited plasmid mini-prep, as well as common issues and troubleshooting. These protocols allow the measurement of reporter proteins in DSM20016, or confirmation via colony PCR if no reporter is involved.

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for ...

Bioengineering

Stable Gene Integration in Chlorella vulgaris Using Agrobacterium tumefaciens

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Author Spotlight: Optimized Transformation Protocol for Chlorella vulgaris Using Agrobacterium tumefaciens 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

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