Bacterial Transformation Workflow

E. coli is the most common bacterial species used in the transformation step of a cloning workflow. In its natural state, the competency of E. coli is very low (10^-5−10^-10) [1] thus cells must be made competent for efficient transformation. The protocols for preparing competent cells vary by choice of transformation method—heat shock or electroporation. Heat shock transformation is also known as chemical transformation and calcium chloride transformation. This method involves subjecting the cells to a sudden increase in temperature, often achieved by briefly immersing them in hot water or placing them in a heating block, followed by a rapid decrease in temperature through incubation on ice. The heat shock causes the cell membrane to become more permeable, facilitating the uptake of exogenous DNA. The alternative terms, chemical transformation and calcium chloride transformation, highlight the different methods and reagents that can be used to enhance the efficiency of DNA uptake during the transformation process. In both cases, a starter culture is prepared by inoculating a single fresh colony from an agar plate into a liquid medium (Figure 2). This starter culture and the subsequent sub-cultures are carefully monitored for active growth by continually measuring optical density at 600 nm (OD₆₀₀). To obtain high transformation efficiency, it is crucial that cell growth is in mid-log phase at the time of harvest—which generally occurs at OD₆₀₀ between 0.4 and 0.9; the optimal value depends on the culture volume, strain, and protocol. Harvested cells are then processed for heat shock treatment or electroporation (Figure 2).

Heat shock transformation: The harvested cells are incubated in calcium chloride (CaCl₂) to make the cell membrane more permeable [2,3]. To further improve competency, Ca²⁺ ions may be supplemented or substituted with other cations and reagents, such as manganese (Mn²⁺), potassium (K⁺), cobalt ([Co(NH₃)₆]³⁺), rubidium (Rb⁺), dimethyl sulfoxide (DMSO), and dithiothreitol (DTT) [4].

Electroporation: The harvested cells are repeatedly washed with ice-cold deionized water by pelleting and resuspension to remove salts and other potential interfering components. After 3–4 wash cycles, the cells are pelleted and resuspended in 10% glycerol for storage [5].

Figure 2. Preparation of chemically competent and electrocompetent cells.

Once prepared, competent cells should be evaluated for transformation efficiency. The transformation efficiency of competent cells is usually measured by the uptake of sub saturating amounts of a supercoiled intact plasmid, approximately 10–500 pg of pUC DNA. The results are expressed as the number of colonies formed (transformants), or colony forming units (CFU), per microgram of plasmid DNA used (CFU/μg) (see Cell Plating). The prepared cells can be stored for future use.

The term “plasmid” was first used by Joshua Lederberg in his 1952 publication and adopted by scientific community to describe a genetic element that is not an organelle, virus, or other cell genetic element. The first plasmid was edited in 1973 demonstrating ability to replace one antibiotic resistant gene (tetracycline) to another (kanamycin) [6]. But the first true vector—plasmid that was used for delivering and manipulating foreign DNA inside a host cell—that became an essential tool in molecular biology was pBR322. Therefore, foreign DNA transfer aided by vectors is called plasmid transformation.

Plasmid uptake by chemically competent cells is facilitated by heat shock, and plasmid uptake by electrocompetent cells is facilitated by electroporation. The choice of method depends on the application, desired transformation efficiency, experimental goals, and available resources; refer to the  Competent Cell Selection Guide. When ready for the transformation step, competent cells should be thawed on ice and handled gently to retain viability. Cells can be mixed by gentle shaking, tapping, or pipetting, but vortexing should be avoided. 

In chemical transformation, plasmid DNA is mixed with chemically competent cells, then briefly exposed to an elevated temperature—a process known as heat shock (Figure 3A). The initial incubation of cells and plasmid is carried out in a polypropylene tube on ice for durations ranging from 5–30 minutes.

For successful chemical transformation, 50–100 µL of competent cells and 1–10 ng of DNA is recommended. When a ligation mixture is used as the source for transforming DNA (often 1–5 µL is sufficient), purification prior to chemical transformation is generally not required. However, ligation mixtures may result in transformation efficiencies as low as 1–10%, compared to transformation with a supercoiled intact plasmid DNA.

Heat shock is performed at 42°C for 30 seconds as appropriate for the bacterial strain and DNA used. For smaller volumes of cells in smaller tubes, the heat shock interval, which depends on the surface-to-volume ratio of the cell suspension, should be reduced. Heat shocked cells are then returned to ice for ≥2 minutes before the next step (Figure 3A).

Figure 3. Bacterial transformation using (A) chemically competent cells and heat shock, and (B) electrocompetent cells and electroporation.

Bacterial transformation aided by electroporation is called electroporation transformation; electroporation involves using an electroporator to subject competent cells and the plasmid carrying DNA construct to a brief pulse of a high-voltage electric field (Figure 3B). This treatment induces transient pores in cell membranes, which permits plasmid entry into the cells (Figure 4). The most common type of electric pulse in bacterial transformation is exponential decay, where a set voltage is applied and allowed to decay over a few milliseconds, called the time constant(Figure 4A). The applied voltage is determined by field strength (V/cm), where V is the initial peak voltage and cm is the measurement of the gap between the electrodes of the cuvette used. Typically, electroporation of bacteria utilizes 0.1 cm cuvettes (20–80 µL volume) and requires a field strength of >15 kV/cm.

Figure 4. Bacterial transformation via electroporation. (A) Graph demonstrating exponential decay of electric pulse over time. (B) Workflow steps of the electroporation process.

One of the main issues with electroporation is arcing, or electric discharge, which may lower cell viability and transformation efficiency. Arcing often results from electroporation in conductive buffers, such as those containing MgCl₂ and phosphates.

After transformation, unused competent cells may be refrozen. However, this will lower transformation efficiencies by about 50% for each freeze/thaw cycle. For best results, aliquot the cells after initial preparation into single-use volumes to minimize freezing and thawing. A single-use format is commercially available to enable transformation and recovery in the same tube and to circumvent the need for freezing and thawing of the cells. To refreeze unused cells, quickly freeze them in a dry ice/ethanol bath for 5 minutes, and store at –70°C. Avoid freezing or storing the cells in liquid nitrogen, which drastically reduces viability.

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Following heat shock or electroporation, transformed cells are cultured in antibiotic-free liquid medium for a brief period. This allows the expression of antibiotic resistance gene(s) from the introduced plasmid (Figure 5). This step improves cell viability and cloning efficiency. For electroporated cells, it is recommended that the cells are grown as soon as possible, since electroporation buffers are not formulated for long-term cell survival.

In the recovery step, prewarmed SOC media is added on the top of transformed cells (250 µL to 1 mL depending on protocol) and cultured at 37°C by shaking (225 rpm) for 1 hour. SOC medium, which contains glucose and MgCl₂, is recommended to maximize transformation efficiency [4]. Use of SOC medium, instead of Lennox L Broth (LB Broth), has been shown to increase 2- to 3-fold formation of transformed colonies [7]. Strains for propagating bacteriophage M13 vectors do not require this step.

Figure 5. Cell growth during the recovery step.

After growing in SOC medium, the cells are plated on solid media, typically LB agar plates with antibiotic(s) or other agents that serve as markers to identify and recover successful transformants. For example, if blue-white screening is performed, X-Gal and IPTG must be included in the agar plate. To ensure that the antibiotic is active, avoid using agar plates more than a few weeks old or days in some cases. Before cell plating, the plates should be prewarmed to a favorable growth temperature and be free of condensation to prevent contamination and mixed colonies.

The number of cells plated should produce enough individual, distinct colonies for further screening, avoiding overly crowded colonies. Cells cultured in SOC medium may be pelleted by centrifugation for 5 minutes at 600–800 x g and resuspended in a smaller volume for plating. For a 100 mm plate, a cell suspension of 100–200 µL is appropriate. If very few or very high number of colonies are present on the agar plate, please follow-up with troubleshooting recommendations.

The culture plates are examined the next day for colony formation. Prolonged incubation should be avoided, as it often results in fusion of large colonies and the appearance of smaller, antibiotic-sensitive surrounding colonies, called satellite colonies, due to antibiotic breakdown around large colonies.

Transformed E. coli colonies then need to be isolated and expanded to isolate and analyze individual constructs. Depending on experimental goal, several or several dozen colonies would need to be picked up and expanded. Usually, colonies are picked up by pipette tip and inoculate in <5 mL LB or TB medium with appropriate selective antibiotic for growth to use for small scale purification (called “miniprep”). If the E. coli strain is growing fast (such as Mach1) then plasmid can be isolated the same day, otherwise minipreps are incubated overnight and high-quality plasmids are isolated using commercial miniprep kits.

Colonies need to be further screened for the presence of the desired DNA construct and to confirm correct sequence. Several approaches can be used to select potential clone with correct insert. First, to confirm if cloning has been successful, vector can be analyzed using restriction enzymes (several DNA fragments of predicted size should be observed) or by PCR using primers flanking insert site (correct length DNA fragment as analyzed in the gel). To be sure that the inserted fragment is correctly assembled (correct direction) or without additional mutations, fragment sequencing is usually performed. Once confirmed, desired colonies may be employed in downstream applications such as plasmid isolation, subcloning, transfection, and protein expression.

Learn more about colony screening methods ›

Measuring performance and preventing procedural errors are key for better experimental outcomes. Click the accordions for helpful suggestions on setting up controls, calculating transformation efficiency, and preventing arcing.

In all steps of bacterial transformation, use sterile tools and labware, media, and reagents as required. It is recommended that once the cells are harvested for further processing, all samples, reagents, and equipment be kept at 0–4°C to improve cell viability and maintain transformation efficiency. Click the accordions for more best practices.

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1. Hasegawa H., Suzuki E., Maeda S. (2018) Horizontal Plasmid Transfer by Transformation in Escherichia coli: Environmental Factors and Possible Mechanisms. Front Microbiol 9:2365. doi: 10.3389/fmicb.2018.02365. 
2. Mandel M and Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53(1):159-162. 
3. Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6(1):23-28.
4. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4):557-580. 
5. Green MR, Sambrook J (2012) Cloning and Transformation with Plasmid Vectors. In: Molecular Cloning: A Laboratory Manual (4th ed). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. pp. 157-260. 
6. Cohen S.N., Chang A.C., Boyer H.W., et al. (1973) Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A 70(11):3240–3244. doi: 10.1073/pnas.70.11.3240. 
7. Invitrogen Corp. (1988) S.O.C. medium for competent cells. Focus 10(3):53.