Transformation of cell
Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA or RNA). In 1928 Frederick Griffith transforms nonpathogenic pneumococcus bacteria into a virulent variety by mixing them in heat-killed pathogenic material. The effect was first demonstrated in 1944 by Oswald Avery, Collin Macleod, and Maclyn McCarty. They showed gene transfer in Streptococcus pneumoniae. Avery. Macleod and McCarty call the uptake and incorporation of DNA by bacteria as transformation. More generally the term is used to describe mechanisms of DNA and RNA transfer in molecular biology. For example the production of transgenic plants like transgenic maize requires the insertion of new genetic information into the maize genome.
Fig: Transformation
Mechanism of transformation
The ability of bacteria to take up foreign DNA is called competence. There are two different forms of competence:Natural competence Natural bacterial transformation occurs only in bacterial species capable of natural competence. Such species carry sets of genes
specifying machinery. It brings DNA across the cell’s membrane or membranes. The evolutionary function of these genes is controversial. Although researchers have assumed that cells take up DNA to acquire new versions of genes. A simpler explanation is that the cells take up DNA mainly as a source of nucleotides. These nucleotides can be used directly or broken down and used for other purposes.
Most naturally transformable bacteria express their competence genes. They develop competence only under specific conditions like response to a nutritional stress. Once the DNA has been brought into the cell’s cytoplasm, it may be degraded by cellular nuclease. But sometimes, it is very similar to the cells own DNA. Therefore, the enzymes that normally repair DNA may recombine it with the chromosome. Natural transformation, is very efficient for linear molecules such as fragments of chromosomal DNA. But it is not used for transfer of circular plasmid DNAs.
Artificial competence
Artificial competence is not encoded in the cell’s genes. Instead it is a laboratory procedure. In this procedure, the cells are passively made permeable to DNA. These procedures are comparatively easy and simple. It can be used to genetically engineer bacteria. There are following methods of artificial competence:
I. The cells are chilled in the presence of divalent cations such as
CaCl2. It prepares the cell walls to become permeable for plasmid DNA. Cells are incubated with the DNA and then briefly heat shocked (42C for 30-120 seconds). It causes the DNA to enter the cell. This method works well for circular plasmid DNAs. But it is not used for linear molecules such as fragments of chromosomal DNA.
- Electroporation is another way to make holes in cells. In this
case, cells are briefly shocked with an electric field of 100-200V. Now plasmid DNA can enter the cell through these holes. Natural membrane-repair mechanisms will close these holes afterwards.
- A plasmid DNA molecule will usually contain an antibiotic resistance gene. It is placed in a bacterial strain that has no antibiotic resistance. Therefore, only transformed bacteria can grow on a media with the antibiotic (this is known as a selection medium). One example of this is putting in a plasmid that contains the encoding for the protein 13-lactamase, whit makes bacteria resistant to ampicillin. This is called the bla gene. The bacterial colony is then treated with ampicillin. It removes those bacteria who did not take tip the plasmid with the bla gene.
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