Choosing the right transformation method and being successful is always tricky.
Choosing between heat shock or electroporation for competent cells will depend on goals, needed efficiency and budget among other things. In the end, trial-and error can help with the long-term decision-making process.
Getting helpful information and tips about transformation methods from experienced researchers is always a good start.
A transformation method induces a bacterium to take up foreign DNA. Although transformation rarely occurs in nature, we can perform artificial transformation in the lab by generating temporary pores on the cell membranes that facilitate DNA intake.
Two standard transformation methods are used in the lab. The first is heat shock transformation, which is based on a combination of chemical elements like calcium or magnesium with temperature changes (cold-heat-cold).
The second approach is electroporation where a device called an electroporator provides an electric pulse over the bacterial cell membranes and create pores.
In this article, I have summarized helpful tips from advanced researchers and my own experience in performing bacterial transformation to help you decide when to choose heat shock or electroporation. Enjoy!
Article Table of Contents
In molecular biology, we say a cell is competent when it has the ability to take up foreign DNA. Therefore, there are two types of competent cells: chemically competent and electrochemically competent (electrocompetent).
Chemically competent cells: are cells that are exposed to elements like calcium chloride (CaCl2) or magnesium chloride (MgCl2) followed by temperature changes from cold (0°C), heat (42°C) and cold again (0°C). This exposure and shock leads to the formation of pores on the cell membrane, allowing cells to take up foreign DNA.
Electrochemically competent cells: are cells that are exposed to an electric pulse and form pores in their membranes facilitating the DNA intake.
To learn more about competent cells, check our GoldBio article Introduction to Competent Cells.
When choosing between heat shock or electroporation, knowing your transformation efficiency needs will be important. In many cases, heat shock transformation (lower cost) will be perfectly acceptable for your experimental goals.
However, certain procedures will require the higher transformation efficiency provided by electroporation.
The range of transformation efficiencies for heat shock is typically around 1 x 105 to 5 x 109 CFU/micrograms, while the range of transformation efficiencies for electroporation is 1 x 1010 to 3 x 1010 CFU/micrograms.
However, the level of transformation efficiency needed will depend on your experimental goals.
Lower efficiencies can be tolerated in applications like
- Routine/ general cloning
- If you have high background colonies
- Tight budget
- Low to high-throughput
- Optimization of transformation methods
High efficiencies will be important when:
- You're producing libraries
- Working with a bigger plasmid
At GoldBio, you may find our Transformation Efficiency Calculator helpful in simplifying the math for your experiments.
Bacterial transformation involves two phases. The first phase involves DNA uptake across the cellular membrane. In the second phase, DNA is set up in the cell as stable genetic material (Asif et al., 2017).
Although the mechanism behind the chemical or electrical transformation is not entirely understood, it is well known that the heat shock method produces a lower transformation efficiency compared to electroporation.
This likely occurs because heat shock hasmore steps which may lead to more errors compared to electroporation.
For instance, cations (Ca2+ and Mg2+) bind to the cell and DNA to neutralize the charge and help anchor DNA to the membrane.
Further, low temperatures congeal the lipid moieties and restrict the cell membrane's fluidity, strengthening the calcium-cell surface interaction
Consequently, high temperatures promote the Brownian motion outside the cell, increasing the probability of pushing DNA inside the cell. The next cold shock restricts the movement of the nonspecific molecules inside the transformed cells.
Brownian motion is the random motion of particles as a result of collisions with their surrounding gaseous or liquid molecules (Big, 2009).
Usually, heat shock requires more DNA than electroporation because heat shock is less efficient. Keep in mind that the amount of DNA is related to the competent cell type.
GoldBio protocols use 1 pg-100 ng of DNA for E. coli chemically competent cells and 1 pg-10 ng of DNA for E. coli electrochemically competent cells.
For Agrobacterium, GoldBio protocols recommend 50 ng to 500 ng of DNA for chemically competent cells and 10 pg-1 ng for electrochemically competent cells.
Before proceeding with transformation protocols, you should check the available resources in your lab.
Although electroporation produces high transformation efficiencies, not all labs have an electroporator. This device produces electric pulses to unbalance the charges in the cell membranes and induce temporary pores to promote DNA uptake.
An electroporator's standard setting for most E. coli strains is 1.8 kV, 25 mF, 200 (Lessard, 2013). However, due to the large variability of competent cells and electroporators available in the market, this setting must be optimized.
Furthermore, other parameters like voltage (V), capacitance (µF), and cell types are also important.
Altogether, the cost range for an electroporator in the market is roughly between $3,000 to $10,000 (Benson, 2020).
- For low transformation efficiency, dilute 1 ml of the overnight culture in 20 ml of LB medium and grow at 37°C with shaking until you reach an OD600 between 0.4 and 0.6 (1–1.5 h). Then use 10 ml to make 50 ml of competent cells.
- Keep your cells on ice at all times. This includes plastic/glass containers and bacteria to ensure high-quality in electroporation. Otherwise, overheating by electric pulses could cause membrane damage.
- If you want to store the electro-competent cells at -80°C for later use, substitute 10% of the distilled water with glycerol and follow the manufacturer's indications.
- During the recovering step, warm up the selective plates to 37°C because cold plates may lead to a lower transformation efficiency.
Below is a glossary of important terms used with often in transformation methods.
The process in which a competent cell is made permeable and can take up foreign DNA.
A plasmid is a small, extrachromosomal DNA molecule within the bacterium that is physically separate from chromosomal DNA and can replicate independently.
DNA fragment with a modified T-DNA region having a foreign gene of interest also called insert.
A cell that has successfully taken up foreign DNA after a transformation process as a result of being exposed to chemicals like calcium or magnesium and temperature changes.
A cell that has successfully taken up foreign DNA after a transformation process as a result of being exposed to electric pulses.
Method where cells take up foreign DNA when they are exposed to chemical solutions with CaCl2 and MgCl2 followed by temperature changes (cold-hot-cold).
Method where cells take up foreign DNA when they are exposed to electric pulses using an electroporator.
Asif, A., Mohsin, H., Tanvir, R., & Rehman, Y. (2017). Revisiting the Mechanisms Involved in Calcium Chloride Induced Bacterial Transformation. Frontiers in Microbiology, 8, 2169. https://doi.org/10.3389/fmicb.2017.02169
Big, C. (2009). Brownian motion. Compendium of Quantum Physics, ISBN : 978-3-540-70622-9.
Froger, A., & Hall, J. E. (2007). Transformation of Plasmid DNA into E. coli Using the Heat Shock Method. Journal of Visualized Experiments, 6, 253. https://doi.org/10.3791/253
Lessard, J. C. (2013). Transformation of E. coli Via Electroporation. En Methods in Enzymology (Vol. 529, pp. 321-327). Elsevier. https://doi.org/10.1016/B978-0-12-418687-3.00027-6
Rahimzadeh, M., Sadeghizadeh, M., Najafi, F., Arab, S., & Mobasheri, H. (2016). Impact of heat shock step on bacterial transformation efficiency. 5.Topp, S. (2009). Comparison of the transformation efficiencies achieved with electroporation vs. Traditional chemical transformation