U3 AOS1 Topic 11: Recombination and Transformation

Recombination is a process by which pieces of DNA are recombined to produce new sequence of DNA.

Transformation is the process by which foreign DNA recombines with the cellular DNA to produce transformed cells.

Bacterial plasmid DNA can be manipulated to produce recombinant plasmids and therefore transform the bacteria. Bacteria’s plasmids can replicate independently from the circular chromosome. Scientists can genetically modify bacteria by incorporating foreign gene of interest into bacterial plasmid DNA to form recombinant plasmids. Bacteria cannot synthesize human proteins therefore the gene of interest for example human insulin gene can be recombined with plasmid DNA so that bacteria can express this gene to make insulin protein by using its own machinery.

Steps of Genetic Modification:

1.  Gene of interest can be isolated from human cells and is amplified using PCR technique.

2.  Plasmid vector is selected which must contain genes coding for some observable traits i.e. antibiotic resistance gene.

3. Restriction endonuclease enzyme is used to cut both the gene of interest and plasmid vector at specific recognition site to create sticky ends. DNA ligase enzyme is used to stick gene of interest with plasmid vector to produce recombinant plasmid.

4.  Recombinant plasmid is taken up by bacteria by the natural process of bacterial transformation. Heat shock and electroporation method can aid this step. The resulting mixture contains both transformed and untransformed bacteria.

5.  The mixture is cultured onto antibiotic-rich plate to carry out antibiotic selection. Untransformed bacteria are killed by antibiotic whereas; transformed bacteria survive as they now contain the recombinant plasmid with antibiotic resistance gene.

6. Colonies of transformed bacteria are now visible on the plate as the single bacterium transformed and multiplied to make its own colony of identical cells.

7. The transformed bacteria are cultured and allowed to produce protein of interest along with bacteria’s own proteins. The protein of interest is the extracted and purified to be used for different purposes.

                            

Importance of bacterial transformation in medical and food industries:

Genetic engineering is more efficient and cheaper method for large scale production of proteins which can be used in food industries for example:

·      Alpha amylase protein for production of ethanol.

·      Chymosin protein for production of cheese.

Proteins for medicine include:

·      Insulin for treatment of diabetes.

·      Growth hormone for growth disorders.

·      Interferon to treat cancer.