Biotech Notes

 

Plasmids

·         Small, circular double stranded DNA molecules in the cytoplasm of many strains of bacteria

·         Plasmid and bacteria have a mutual arrangement: Plasmid uses the enzymes and ribosome of the bacterial cell, while bacterial cells benefit from the genes carried and expressed by plasmids to confer antibiotic resistance. The relationship between bacteria and plasmids is endosymbiotic.

·         Higher copy number means for more copies of a plasmid existing in a host bacterial cell, thus more protein will be synthesized to strengthen the resistance to antibiotics.

·         Multiple cloning is a region in an artificial plasmid where it has been engineered to contain many recognition sites of various restriction endonucleases.

·         If the foreign gene has been excised using the same restriction enzyme, it will anneal and permanently become part of the plasmid. DNA ligase then reforms the phophodiester bonds, and now the plasmid is recombinant DNA. As this plasmid replicates, copies of the recombinant DNA are produced, and this gene is said to be cloned.

Transformation:

·         Introducing DNA from another source is called transformation, and vectors such as plasmid, carry a desired gene into a host cell.

·         Competent cells readily take up foreign DNA. Cells that are not naturally competent can be chemically induced with the aid of calcium chloride. As the positive calcium ions stabilize the negative charges of the phosphates (membrane), the solution is quickly heated that creates a draft. This draft sweeps the plasmids into the cell through the membrane.

·         It is possible that the bacteria exhibit antibiotic resistance by taking up the plasmid that has failed to transform. Therefore, it is necessary to check that the gene exists in the transformed bacteria. This is again accomplished by using a restriction enzyme to release the cloned fragment from the vector after colonies of growth.

·         Selective plating isolates the cells with recombinant DNA. If transformation is successful, the bacteria will be able to resist the antibiotic.

·         Today, electroporators are also used, which subject the bacteria to an electric shock that loosens the structure of the cell membrane to allow foreign DNA to enter.