Structural Biochemistry/Bacterial transformation technique
Bacterial Transformation
[edit | edit source]Bacterial Transformation is a basic technique that is commonly used in a molecular biological laboratory. The technique is performed by introducing a foreign plasmid into the bacteria and the bacteria is used to amplify the foreign DNA. Bacteria can replicate the foreign DNA if the foreign DNA has an origin of replication that its DNA polymerases can recognize. The bacteria will then replicate the foreign DNA along with its own. This new DNA strand can at times provide the organism with new trait(s) that can be identified after transformation.
Role of Heat Shock in Transformation
[edit | edit source]Heat Shock is subjecting a cell to a higher temperature than it is normally found in the organism. Heat Shock causes pores to form in the outer membrane which permits DNA to enter the cell. The heat source is then removed from the cell and the membrane reforms with the DNA inside it.
Role of Electroporation in Transformation
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The role of electroporation in transformation is the same as Heat Shock, though the method is different. In electroporation, an electric field is applied to the cell that has a significant increase in the electrical conductivity and permeability of the cell membrane which allows foreign DNA to enter the cell. Bacteria also contain a cell wall made of peptidoglycan that are naturally porous and only act as a shell that protects the bacteria. If plasmids are mixed with bacteria, plasmids can be transferred into the cell after electroporation. The electric field in electroporation is created by appliances called electroporators.
Role of Calcium Chloride in Transformation
[edit | edit source]The addition of Calcium Chloride produces calcium ions which help the cell membrane become more permeable to DNA plasmid. This method of disrupting the cell membrane using electronics is generally used in conjunction to heat shocking. In which drastic changes in temperature causes the membrane to be more porous, enabling a greater efficiency for the desired DNA segments to transform into the targeted bacteria. This method of competence is more effective when used with plasmid DNA than linear DNA since certain enzymes exist within the bacteria that readily degrade linear DNA.