Bacterial Transformation and Transformation Protocol

Organisms must adapt to ever-changing environments, otherwise they risk being outcompeted or eliminated. To do this, they have evolved methods for importing DNA from elsewhere and utilizing it for their own purposes, transformation being one such method.

In this article, we will discuss transformation, bacterial transformation in particular, and how scientists can utilize it to manipulate bacteria for a plethora of purposes.

What is transformation in biology?

Transformation is the process by which organisms take up foreign genetic material directly from their surrounding environment and incorporate it into their genome by homologous recombination or reconvert it into extrachromosomal elements like plasmids. It is one of the three methods that lead to horizontal gene transfer (HGT), along with conjugation and transduction. Natural transformation is observed in certain eukaryotic systems, including fungi¹ and plants.² Artificial methods like electroporation, gene gun and microinjection have also been developed and extensively used by scientists to manipulate and study eukaryotes, but for this article, we will concentrate on bacterial systems.

What is bacterial transformation?

Bacterial transformation was discovered in Streptococcus pneumoniae (S. pneumoniae) by Frederick Griffith in 1928.³ He was conducting vaccination experiments in mice with S. pneumoniae bacteria and found that non-virulent bacteria could be transformed into virulent bacteria by adding heat-killed virulent bacteria. This “transforming principle” was later identified as DNA by Oswald Avery, Colin MacLeod and Maclyn McCarty, and they coined the term transformation.⁴

Transformation of bacteria can occur in two distinct ways: naturally or artificially. As the name suggests, natural transformation can occur naturally but can also be induced under laboratory conditions. Unlike transduction and conjugation, transformation is entirely driven by the recipient. In most species, and under certain stressful environmental conditions like starvation, competence genes encoded in their core genome are transiently expressed, producing the required proteins to become “competent” for transformation. Natural transformation has been demonstrated in more than 80 bacterial species, ranging from very well-understood Gram positive species, e.g., Bacillus subtilis and S. pneumoniae, and Gram negative species, e.g., Haemphilus influenzae and Vibrio cholerae, to the more obscure species that most people will never have heard of.⁵

Artificial bacterial transformation is a laboratory technique scientists use to introduce foreign DNA into bacterial cells. There are two main methods by which this is achieved:

• Electroporation: In this method, an electrical pulse is delivered (by an electroporator) across a bacterial cell suspension containing the DNA of interest, which generates small pores through which DNA can enter.
• Heat shock transformation: In this method, bacteria are made “competent” by chemical means, like calcium chloride (CaCal₂), and are then subjected to a heat shock that transfers the DNA of interest into the cell through small pores.

Bacterial transformation steps

Natural transformation

Gram positive and Gram negative organisms rely on conserved com regulons, which are involved in the uptake and processing of exogenous double-stranded DNA (dsDNA).

1. Initial binding of dsDNA is done through a pilus structure called the transformation pilus (Tfp), which projects into the extracellular medium (Figure 1A).
2. Once bound, the dsDNA is transferred to a receptor and then to an enzyme that processes the DNA into a single-stranded molecule (ssDNA, Figure 1B)
3. The ssDNA passes through a membrane-bound pore driven by ATP. The only significant difference between Gram positive and Gram negative organisms is the requirement for an additional channel to transfer dsDNA across the outer membrane of Gram negative species.
4. The internalized ssDNA is loaded onto a recombinase by a transformation processing protein (Figure 1C), which then proceeds with a homology search of the genome. Once a homologous region is found, recombination is initiated; if not, the DNA will be degraded.^5,6

Figure 1: A diagram showing the steps of natural transformation. A) Binding of dsDNA, B) Transport and conversion into ssDNA, C) Binding of transformation processing protein. Credit: Technology Networks.

Artificial transformation

In both natural and artificial transformation, the steps to obtain transformed bacteria are essentially the same but through different means. In artificial transformation (Figure 2), competent cells are generated artificially (Figure 2A), e.g., with CaCl₂ for chemically competent cells or repeated washing in an inert sugar, like sucrose, for electrocompetent cells. Please note that all of the steps in producing either type, once the initial bacterial cells have been grown (normally at 37 ^ºC) are done on ice as the cells become quite delicate. The DNA of interest (often a plasmid containing an antibiotic resistance marker) is then added to the competent cells, which are then either heat shocked (in the case of chemically competent cells) or have an electrical current passed through them (in the case of electrocompetent cells) (Figure 2B). The cells then have ice-cold media added to them. They are then transferred back to 37 ºC to allow recovery (Figure 2C) before they are plated onto selective agar plates containing the appropriate antibiotic. These are then incubated to allow transformed cells to form colonies (Figure 2D).

Figure 2: The steps to generating artificially transformed cells. A) Generate competent cells, B) Transform competent cells, C) Allow recovery after transformation, D) Select for transformants. Credit: Technology Networks.

An example bacterial transformation protocol

Let’s consider an example of a bacterial transformation protocol using chemically competent cells and heat shock.

1. A single colony of Escherichia coli is transferred from an agar plate to a rich liquid medium and grown overnight at 37 ºC.
2. The culture is diluted 1 in 10 and grown until the mid-logarithmic phase is reached (OD₆₀₀ nm of 0.6).
3. The cells are chilled on ice.
4. Centrifugation is used to harvest the cells, and the supernatant is discarded.
5. The cells are re-suspended in ice-cold 0.1 M CaCal₂ for 30 minutes; they are now competent for transformation.
6. The DNA to be transformed into the cells (normally a plasmid with an antibiotic selection marker) is added to an aliquot of competent cells (typically 100 µl). The solution is incubated on ice for 30 minutes.
7. The cell/plasmid solution is heat shocked at 42 ºC for 90 seconds.
8. The cell/plasmid solution is cooled back to 4 ºC for 3 minutes.
9. Ice-cold rich media is added to the cells, and the mixture is incubated at 37 ºC for 60 minutes.
10. Aliquots are plated onto selective agar plates containing the antibiotic appropriate for the plasmid used to transform the cells.
11. The plates are incubated overnight at 37 ºC and colonies that have been transformed, and thus contain the antibiotic resistance gene too, will be visible.

Transfection vs transformation

Both transfection and transformation are used to introduce foreign DNA into cells; however they differ in their context, applications and the type of cells involved, as summarized in Table 1.

Table 1: A comparison between transfection and transformation.

How is artificial transformation in bacteria harnessed in science?

Artificial transformation in bacteria is a powerful tool in molecular biology and biotechnology. There are multiple applications, including but certainly not limited to cloning, protein production for numerous applications,^7,8 CRISPR-Cas9 gene editing,⁹ genetic engineering to study gene function,¹⁰ synthetic biology¹¹ and the construction of DNA libraries for a variety of purposes including drug discovery¹² and bioremediation.¹³