Question

what is transformation and selection?

what is the descriptive process of selection and transformation?

Answer 1

Transformation is a technique in molecular biology and genetics, where in there is a  genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane. For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory. Transformation is a key step in DNA cloning. It occurs after restriction digest and ligation and transfers newly made plasmids to bacteria.

After transformation, the next process in recombinant DNA technology is the selection of bacteria with the desired gene of interest which has been transformed with a vector in a host cell. Both transformation and selection are essential steps in DNA cloning.

After transformation, bacteria are selected with antibiotic plates. Bacteria having the intact plasmid for an antibiotic - resistance gene are considered to be antibiotic resistant and will form a colony on the plate. Colonies with the right plasmid are taken from primary cultures and are further subcultured to make large cultures of identical bacteria, which are used to produce plasmid or make protein.

In a typical cloning experiment, researchers first insert a piece of DNA, such as a gene, into a circular piece of DNA called a plasmid. This step uses restriction enzymes and DNA ligase and is called a ligation

After a ligation, the next step is to transfer the DNA into bacteria in a process called transformation. Then, we can use antibiotic selection and DNA analysis methods to identify bacteria that contain the plasmid we’re looking for.

Selection is necessary to identify colonies containing the gene which we are in need of i.e - antibiotic resistance gene. During several instances, it might be that when we try to insert a gene into a plasmid using a particular restriction enzyme, we may get some cases where the plasmid closes back up (without taking in the gene), and other cases where the gene goes in backwards. In that case DNA transcription is hampered and no product will be formed. Hence due to such discrepancies, it is necessary to collect plasmid DNA from each colony and check to see if it matches the plasmid we were trying to build and select those colonies expressing the plasmid in the right direction so that the promoter is activated and genetic transcription takes place, thus conferring resistance to the concerned antibiotic.

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