transformation of bacteria

Bacterial transformation consists of a bacteria cell uptaking a preprogrammed plasmid or cosmid. The plasmid consists of a vector that always contains a segment of DNA that expresses antibiotic resistance. This is both beneficial to the bacteria that uptakes the plasmid and also provides a means of identifying and isolating the cells that have successfully uptaken the desired plasmid. The vector plasmid is usually cleaved at one of the MCS (Multiple Cloning Site) by a unique restriction endonuclease and then a piece of DNA is inserted. Vector and DNA ligate together to form a new plasmid now expressing the newly inserted DNA (hopefully in the right direction) as well as the antibiotic resistance and any additional gene expression encoded in the vector.

A general protocol for bacterial transformation involves heat shocking and ice shocking the bacteria alternately. Note: You can also use Ca(2+) or electrophoretic waves, but I find this to be easiest to use. An example of heat/ice shocking transformation protocol is as follows:

1. Fetch a tube of frozen bacterial cells (various strains of e. coli are most popular) from the -80C refrigerator.

2. At your bench, warm the tube with your fingers until the cells just begin to thaw and place on ice.

3. Add 10 ul of plasmid DNA to approximately 100 ul of bacteria (if the DNA is not supercoiled).

4. Mix by briefly by finger flicking tube then incubate on ice for 30 min.

5. Heat shock the cells by placing the tube in a 37C incubator or water bath for 45 seconds and return to ice.

6. After 2-3 min, add 0.5 ml LB (Luria-Bertani) broth without antibiotics, mix, and incubate at 37 C for 1 hr with shaking.

7. After incubation, shake tube and aliquot 100 ul (or as much as 500 ul) of the suspension into the middle of an LB/ampicillin (use the appropriate antibiotic depending on the vector used) plate as a puddle.

8. Spread by evenly spreading cells over the plate using a “hockey puck” spreader or giant tooth pick. Be sure that you have dipped your spreader in ethanol and flamed it before spreading or that the tooth pick has been autoclaved.

9. Incubate at 37C overnight with the agar side up and the lid side down (don't forget to mark the plate properly).

The next day you should see nice little seperate colonies and then you will be ready to pick clones and see which colonies have the correct insert! What excitement!!

There are many scientific protocols for the tranformation of bacteria, and indeed, it forms a cornerstone of molecular biology, along with PCR, gel electrophoresis, and restriction enzymes.

But the fact that bacteria can so easily take up exogenous DNA points to the fact that they use rapid mutation as a way to cope with their environment. Not only do they swap DNA within a given species through carefully choreographed methods (conjugation via pili), but they take up totally foreign DNA to boot. They even have enzymes that facilitate integration of said DNA into their chromosomes.

This is also one reason why the rampant over-use of antibiotics is dangerous. They lead to the development of antibiotic-resistant bacteria, and once a mutation leading to drug-resistance happens, the DNA containing the gene can spread to other bacteria found within the host.

Bacteria and viruses are sneaky bastards.

A molecular biology technique where foreign DNA is introduced into bacterial or yeast cells. This method is analogous to transfection, where the DNA is instead inserted into eukaryotic cells. Both techniques use DNA that typically encodes a gene of interest. This DNA is placed into a plasmid before transformation. Transformation is thought to be first demonstrated in the bacteria Streptococcus pneumoniae by Frederick Griffith in 1928. His famous experiment helped to prove that DNA, not protein, was the unit of hereditary information in the cell.

There are two main reasons to transform cells:

1. To produce a large amount of the DNA. The bacteria are used as a host to grow a high number of plasmids. The plasmids can then be isolated from the bacteria to obtain a large amount of purified DNA. The DNA can then be used for sequencing, transfection, or other techniques that require pure DNA.
2. To produce a large amount of protein from the DNA. In this case the DNA of interest is a gene that encodes a protein product. If the proper plasmid is used the bacteria can be induced by a compound called IPTG to start expressing the protein. The protein can then be isolated for use in other techniques.

Unlike eukaryotic cells, bacterial cells first require an extra step to encourage them to take up the DNA. This is called making the bacteria competent. The bacteria are placed in a calcium chloride solution and the chloride ions from the solution are able to pass through the bacterial cell membrane. These ions also bring water molecules with them, which causes the cell to swell. This swelling encourages the bacteria to take up foreign DNA, although the exact reason why is unknown. Biotechnology companies sell bacteria cells that are already competent, allowing researchers to skip this step.

Cells can be transformed by using one of two techniques:

1. Heat shock
   First, plasmid DNA is mixed with the bacteria cells and placed on ice. The cold temperature is thought to help the DNA adhere to the cell membrane. Next, the bacteria are "shocked" by abruptly increasing the temperature. This is done by placing them in a warm (42°C) water bath for a short period of time. The shocked bacteria are more likely to take up DNA. Exact temperature is important to obtain the highest transformation efficiency. If the temperature is too cold the bacteria will not take up the DNA, while if it is too hot the bacteria will die. This method is less efficient than electroporation, meaning fewer bacteria will take up the DNA. However, it is quick, easy, and does not require an electroporator machine.
2. Electroporation
   The bacteria and DNA are mixed together and subjected to a high voltage of electricity. This creates holes in the cell membrane, allowing the DNA to enter the cell. The exact voltage varies rather widely between different strains of bacteria. Electroporation is also used for transfection, although the voltage required is much lower than with transformation. This method is the more efficient than heat shock, but requires a special, expensive device called an electroporator.

After either of these methods are performed it is necessary to let the bacteria recover from the shock. This is done by placing them in a medium such as LB medium and shaking them at a warm temperature for about an hour. In order to increase DNA or protein yields, bacteria that have been transformed are often isolated from untransformed cells by treating the bacteria with an antibiotic. The plasmid DNA often contains a gene whose encoded protein renders the bacteria immune to the antibiotic. Therefore, only bacteria that have been transformed will be able to survive.

For more reading:

• http://www.genome.ou.edu/protocol_book/protocol_adxF.html
• Current Protocols in Molecular Biology, Volumes 1 and 2, 1998