CaCl2 Transformation Technique

Introduction

Competent cells are bacterial cells that can accept extra-chromosomal DNA or plasmids (naked DNA) from the environment. The generation of competent cells may occur by two methods: natural competence and artificial competence. Natural competence is the genetic ability of a bacterium to receive environmental DNA under natural or in vitro conditions. Bacteria can also be made competent artificially by chemical treatment and heat shock to make them transiently permeable to DNA. Natural competence dates back to 1928 when Frederick Griffith discovered that prepared heat-killed cells of a pathogenic bacterium could transform the nonpathogenic cells into pathogenic type. Natural competence has been reported in many bacterial strains, i.e. Bacillus subtilis, Streptococcus pneumonia, Neisseria gonorrhoeae and Haemophilus influenza. The natural competence phenomenon is highly regulated in bacteria and varies across genera. In some genera, certain portions of the population are competent at a time, and in others, the whole population gains competence at the same time. When the foreign DNA enters inside the cells, it may be degraded by the cellular nucleases or may recombine with the cellular chromosome. However, natural competence and transformation are efficient for linear molecules such as chromosomal DNA but not for circular plasmid molecules. Artificial competence is not coded by the genes of the bacterial cells. It is a laboratory procedure in which cells are passively made permeable to DNA using unnatural conditions. The procedure of artificial competence is relatively simple and easy and can be used to engineer a bacterium genetically. However, transformation efficiency is very low as only a portion of the cells become competent to successfully take up DNA.

Principle

As DNA is a highly hydrophilic molecule, normally it cannot pass through the cell membrane of bacteria. Hence, in order to make bacteria capable of internalizing the genetic material, they must be made competent to take up the DNA. This can be achieved by making small holes in bacterial cells by suspending them in a solution containing a high concentration of calcium. Extra-chromosomal DNA will be forced to enter the cell by incubating the competent cells and the DNA together on ice followed by a brief heat shock that causes the bacteria to take up the DNA. Bacteria no longer become stable when they possess holes on the cell membrane and may die easily. Additionally, a poorly performed procedure may lead to not enough competence cells to take up DNA. It has been reported that a naked DNA molecule is bound to the lipopolysaccharide(LPS) receptor molecules on the competent cell surface. The divalent cations generate coordination complexes with the negatively charged DNA molecules and LPS. DNA, being a larger molecule, cannot itself cross the cell membrane to enter into the cytosol. The heat shock step strongly depolarizes the cell membrane of CaCl2-treated cells. Thus, the decrease in membrane potential lowers the negativity of the cell’s inside potential which ultimately allows the movement of negatively charged DNA into the cell’s interior. The subsequent cold shock again raises the membrane potential to its original value.

Competent cells are ready to use bacterial cells that possess more easily altered cell walls by which foreign DNA can be passed through easily. Most types of cells cannot take up DNA efficiently unless they have been exposed to special chemical or electrical treatments to make them competent. The standard method for making the bacteria permeable to DNA involves treatment with calcium ions. Brief exposure of cells to an electric field also allows the bacteria to take up DNA and this process is called as electroporation. However, some types of bacteria are naturally transformable, which means they can take up DNA from their environment without requiring special treatment. The exact mechanisms involved in artificial competence are not yet known well. ln CaCl2 method, the competency can be obtained by creating pores in bacterial cells by suspending them in a solution containing a high concentration of calcium. DNA can then be forced into the Host cell by heat shock treatment at 42oC for the process of transformation

Competence is distinguished into natural competence, a genetically specified ability of bacteria that is thought to occur under natural conditions as well as in the laboratory and induced or artificial competence, arising when cells in laboratory cultures are treated to make them transiently permeable to DNA.

Natural Competence

Bacteria are able to take up DNA from their environment by three ways; conjugation, transformation, and transduction.  In transformation, the DNA is directly entered into the cell.  Uptake of transforming DNA requires the recipient cells to be in a specialized physiological state called competent state. Natural competence was first discovered by Frederich Griffith in 1928. It is highly regulated in bacteria, and the factors involved in competence vary among genera. The competence proteins produced have some homology but differ in the Gram-negative and the Gram-positive bacteria.  Once the DNA has been brought into the cell’s cytoplasm, it may be degraded by the nuclease enzymes, or, if it is very similar to the cells own DNA, the DNA repairing enzymes may recombine it with the chromosome.

Artificial Competence and Transformation

Artificial competence is not encoded in the cell’s genes. Instead, it is a laboratory procedure by which cells are made permeable to DNA, with conditions that do not normally occur in nature. This procedure is comparatively easy and simple, and can be used in the genetic engineering of bacteria but in general transformation efficiency is low.  Methods for preparing the competent cells derive from the work of Mandel and Higa who developed a simple treatment based on soaking the cells in cold CaCl2. There are two main methods for the preparation of competent cells.They are Calcium chloride method and Electroporation.

Rapidly growing cells are made competent more easily than cells in other Growth stages. So it is necessary to bring cells into log phase before the procedure is begun. The cells in rapid growth (log phase) are living, healthy, and actively metabolizing. Competent cells are readily available in commercial markets.

Reagents Required and Their Role Luria-Bertani Broth

Luria-Bertani (LB) broth is a rich medium that permits fast growth and good growth yields for many species including E. coli. It is the most commonly used medium in microbiology and molecular biology studies for E. coli cell cultures. Easy preparation, fast growth of most E. coli strains, ready availability and simple compositions contribute to the popularity of LB broth. LB can support E. coli growth (OD600 = 2–3) under normal shaking incubation conditions.

Calcium Chloride

Calcium chloride transformation technique is the most efficient technique among the competent cell preparation protocols. It increases the bacterial cell’s ability to incorporate plasmid DNA, facilitating genetic transformation. Addition of calcium chloride to the cell suspension allows the binding of plasmid DNA to LPS. Thus, both the negatively charged DNA backbone and LPS come together and when heat shock is provided, plasmid DNA passes into the bacterial cell. Prepare 2000 ml of 50 mM Calcium

chloride stock solution by adding 14.701 g of CaCl2.2H2O in 2 l of milli-Q water, autoclave, and store at 4 °C.

Materials

  1. LB broth: Yeast extract 0.5%, NaCl l%, tryptone 1%.

2 LB agar: As above, plus 2% agar prior to autoclaving.

  1. 0.1M CaCl2.

Antibiotics are added to the above media after autoclaving. Tetracycline to a final concentration of 15 pg/mL and ampicillin to 50 kg/mL Solutions of these antibiotics are prepared with ampicillin at 50 mg/mL m slightly alkaline distilled water and tetracycline at 15 mg/mL in ethanol.

Method

  1. Prepare a small, overnight culture of the bacteria in LB broth. Grow at 37°C without shaking.
  2. About 2 h before you are ready to begin the main procedure, use 1.0 mL of the overnight culture to inoculate 100 mL of fresh LB broth. This culture is grown with rapid shaking at 37°C until it reaches roughly 5 x 107 cells/ml. Thus corresponds to an OD650 for our cultures, but you should calibrate this for each of your own strains
  3. Take a 5 mL aliquot of each transformation reaction and transfer to sterile plastic centrifuge tubes. Cool on ice for 10 mm.
  4. Pellet the cells by spinning for 5 mm at 5000g. It is necessary for the centrifugation to be performed at 4°C. We have found a refrigerated bench centrifuge ideal for this.
  5. Pour off the supernatant and resuspend cells in 25 mL of cold 0.1M CaCl2. Leave on ice for at least 20 min.
  6. Centrifuge as in Step 3. You should observe a more diffuse pellet than previously. This is an indication of competent cells.
  7. Resuspend the cells in 0.2 mL of cold 0.1M CaCl2.
  8. Transfer the suspensions to sterile, thin-walled glass bottles or tubes. The use of glass makes the subsequent heat shocks more effective.
  9. To each tube add up to 0.1 mg of DNA, made up in a standard DNA storage buffer such as TE to a volume of 100 mL. Leave on ice for 30 min.
  10. Transfer to a 42°C water bath for 2 min and return briefly to ice.
  11. Transfer the contents of each tube to 2 mL of LB broth in a small flask. Incubate with shaking at 37°C for 60-90 min.
  12. Plate 0.1 mL aliquots of undiluted, 10-1 and 10-2 dilutions onto LB plates to which the antibiotics to be used for selection have been added.
  13. Incubate overnight at 37°C.

Notes

  1. This method generally gives 104-106 transformants/mg of closed circle plasmid DNA. Do note that the relationship between amounts of DNA added and yield is not totally linear. Greater than 0.1 mg of plasmid DNA per tube will decrease transformation efficiency.
  2. It is essential that the cells used are in a rapid growth phase when harvested. Do not let them approach stationary phase.
  3. Cells can be stored at 4°C once competent. Holding cells in CaCl2 at 4°C will, in fact, increase transformation efficiency although this declines with more than 24 h storage. Long periods of storage can be achieved by freezing the competent cells.
  4. The revival step is necessary both to allow the plasmid establishment and to allow expression of the resistance genes.
  5. One problem encountered on plating on ampicillin is that resistant colonies will often be surrounded by a region of secondary growth. This is caused by the p-lactamase activity of the resistant cells hydrolyzing the surrounding antibiotic and thus allowing surviving sensitive cells to begin to grow. This problem can be avoided by using freshly made ampicillin plates and removing plates from the incubator promptly after the period of overnight growth.