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Home >> Generation of Clones - Generation of Insert >> Transformation and Multiplication of Clones
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Transformation and Multiplication of Clones - The central step in a gene cloning procedure is to transfer a recombinant clone generated in vitro, into bacteria or any other host. The concept and feasibility of molecular cloning is centred around two principles. Ligation in vitro generally yields a population of DNA molecules out of which only some are important.

Hence the transformation step should ensure that one cell receives a single plasmid or molecule. This results in separation of each recombination from all the others. Each recipient cell needs to separate from all the others in the population to permit isolation of a clone of cells.

Isolation of a clone of cells depends upon the property of the host-vector combination being used. Before transformation we have to keep the host vector combination and screening methods in mind and select the host, as the host properties are very important in transformation and multiplication.

In most of the cases, microorganisms which are proved unlikely to survive in nature are used so that accidental release of genetically modified strains can be prevented.

Today, hundreds of such microorganisms are available. Hence when selecting a host for transformation, three important points must be kept in mind. Firstly the host must supply the factor required for the vector replication and should not contain any elements that inhibit vector multiplication or prevent some screening methods being used.

Secondly, the cell should not contain any active restriction enzymes being synthesized, as this will cleave the recombinant molecule. Even the host cells which provide E. coli dam and dam methyl transferase are avoided, as they will generate replicated recombinant molecules which cannot be cleaved by some restriction enzymes due to methylation of the recognition site.

Lastly, the host should not have any phenotypic character which is similar to the vector. For example, if the vector codes for ampicillin resistance, the host must be susceptible to ampicillin antibiotic in the absence of vector. As a general rule, the host cell should be sensitive to particular antibiotics or toxins and should not harbour any extraneous plasmid (e.g. F+ E. coli cells).

After the host is decided, the second step is deciding the approach to introduce the recombinant DNA molecule. Normally three different approaches have gained wide popularity and acceptance. The first one is uptake of plasmid by chemically treated cells.

Most species of bacteria including E. coli take up only limited amounts of DNA under normal conditions and have to undergo a chemical pre treatment before they can be transformed efficiently. Cells which have undergone such a treatment are said to be competent cells.

Mandel and Higa developed this process in 1970. In this method, mid-log phase E. coli cells are taken and washed with 0.05 M CaCl2 twice, to remove the traces of growth medium. Then Ca++ ions bind to the membrane with positive charge. Now as the membrane is surrounded by Ca++ ions, DNA molecules bind to them due to the nature of DNA molecule.

Now Ca++ ions act as a bridge between the membrane and DNA molecule. When cells are subjected to the heat shock in the presence of recombinant DNA molecules, Le., when the cells are exposed to 42°C for 45-55 seconds, the membrane of the bacteria slightly expands, the negative charge gets weakened and holes are created in the membrane, thus creating a pathway for the migration of the DNA and Ca++ ions into the cell.

Once inside the cell, the Ca++ ions are expelled out. Of course the biological basis of the techniques is not yet understood. This is the explanation widely accepted. This method is very reliable and works well with most strains of E. coli Recombinant cells prepared by this method yield more than satisfactory results for most standard cloning procedures.

The other advantage of this method is the ability to retain high transformation efficiency even when cells stored for months at -80°C are used. The only drawback of this method is lower transformation efficiency, which is not sufficient when the aim is construction of the genomic library.

This drawback has lead to the development of a sophisticated method where an electrical shock is given to the cells instead of heat shock. This method is called as electroporation.

In electroporation, the cells are exposed to an electric field of 12 kV/cm2 for about a period of5-10 milliseconds. This shock induces holes in the bacterial cell membrane through which the DNA enters before the holes are repaired. Transformation efficiency frequency will be 100-1000 times greater than that of the chemical method.

The only drawback in this method is the requirement of washing the cells repeatedly with water prior to electroporation because traces of metals in the bacterial suspension can cause total explosion of the instrument or a heavy reduction in transformation efficiency.

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