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Principles and Techniques Behind Bacterial Transformation

BY: Lakshmi K Sugavanam | Category: Biotech-Research | Submitted: 2011-04-10 01:21:17
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Article Summary: "The techniques employed in the insertion of plasmids into bacterial cells. Transformation was demonstrated by Frederick Griffith in 1928 when he discovered that a non-virulent strain of Streptococcus pneumoniae converted to a virulent form after exposure to heat-killed virulent strains..."

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Transformation was demonstrated by Frederick Griffith in 1928 when he discovered that a non-virulent strain of Streptococcus pneumoniae converted to a virulent form after exposure to heat-killed virulent strains. He held a 'transforming principle' from that virulent strain responsible for this change. Then, in 1944, Avery, MacLeod and McCarty identified the genetic nature of this transforming principle by isolating the genetic material from the virulent Streptococcus and used it to make a non-virulent strain virulent. They called this phenomenon of uptake of virulent DNA and its expression as transformation. Transformation occurs most commonly in bacteria. It can also be performed by artificial means.

Electroporation as an aid to transformation was introduced in the late 1980s which improved the rate of transformation. Other methods like micro injection and particle gun method are also commonly used methods of transformation. Today, transformation is performed on a daily basis in molecular biology laboratories to introduce foreign plasmids into bacteria for amplification purposes.

The plasmid used in transformation is a small piece of circular DNA that can range from 2kb to10kb in size. They carry some crucial genes essential for the growth of the bacteria. These include antibiotic resistance genes. Soon after the discovery of plasmids, it was realized that they could be used as vehicles for the process of cloning genes of interest. So, a plasmid with at least one antibiotic resistance gene, which acts as a selective marker, is used as a vector. The gene of interest is then inserted into this plasmid then this vector is inserted into E.coli sensitive to ampicillin.

The mixture may contain both transformed and non-transformed E.coli cells. So, in order to select transformed cells, the mixture is then spread on culture plates that contain ampicillin which acts as a selective pressure. Only the transformed E.coli can grow and form colonies. These will also contain the gene of interest. Another method of selection is blue-white screening that involves the lacZ gene.

These plasmids will also contain an origin of replication which is used by the replication machinery during DNA replication as a starting point, a multiple cloning site that will contain the restriction sites for multiple restriction enzymes where the DNA of interest can be inserted. The multiple cloning site is usually located in the middle of a reporter gene sequence. The various commercial plasmids available vary with respect to the kind of restriction sites, their position, the type of antibiotic they are resistant to etc.

The hydrophilic nature of DNA will prevent it from passing through the bacterial cell membrane. In order for the target DNA to enter the cell, the bacterial cell membrane has to be made passively permeable to DNA. This is done by creating temporary holes in the cell membrane by various methods. One is by suspending the bacterial cells in a high concentration of calcium chloride placed on ice. Then, a heat shock is given to the ice cold mixture which allows the DNA to enter the cells and then it is replaced on ice. This allows the transformation to occur. It works especially well for circular plasmids. Good preparations should easily give 105 to 106 transformants per microgram of plasmid. This method may not well for linear DNA, because the native exonucleases degrade the linear DNA. However, cells that are naturally competent are transformed better with linear DNA than with plasmid DNA.

Another method is sonication where the cells are placed in a sonicator and the pores are created by ultrasound vibrations. Electroporesis may also be used to for the same purpose. The cells are given a brief shock with an electric field of about 10-20 kV/cm which creates holes in the membranes that can take up large plasmids. Then the holes are repaired by the cell's natural repair mechanisms. When the membrane is able to allow the DNA to be taken up, it is said to be a 'competent cell'. Transformation must always be performed under sterile conditions as it is possible to denature the plasmid or contaminate the mixture easily. The efficiency with which the foreign DNA is exposed is called as transformation efficiency.

Transformation may also be performed on plant or animal cells, although in the case of eukaryotic cells, it is referred to as transfection. DNA can also be transferred between strains by a process called horizontal gene transfer.

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