Transformation
I. Introduction
The purpose of the lab is to calculate the transformation efficiency of bacteria exposed to plasmid conferring antibiotic Ampicillin resistance and bioluminescence.
Transformation is the uptake of foreign DNA by competent cells to express a foreign gene. In order to be competent, the cell must be in logarithmic growth. It also must have a weak cell wall and plasma membrane. The cell then should undergo rapid binary fission. The foreign DNA is transferred to the competent cell via a vector. The primary vector used is the plasmid. Plasmids are extra chromosomal and contain the gene of interest, in this case bioluminescence. The R plasmid, which causes the resistance to Ampicillin, confirms antibiotic resistance. It makes it able to identify which cells under went transformation. Restriction endonucleases can be used to cut and insert pieces of foreign DNA into the plasmid. Once the bacteria or competent cell takes up the plasmid, the restriction enzymes in the host cell immediately begin cutting up the foreign DNA. The DNA can then fit in the host DNA due to sticky ends. Once the host cell accepts the plasmid, it has been transformed. The transformation efficiency can then be calculated by the following calculation:
Number of transformants * final volume at recovery (ml) = number of mg of DNA volume plated (ml) transformants per mg
Those cells that accept the plasmid will be identified by growing on an Ampicillin positive plate and glow in the dark.
If the exposure of bacteria to plasmid pAMP/pBlu is increased, then the transformation efficiency will increase.
The dependent variable is the rate of transformation efficiency as measured by number of colonies glowing.
The independent variable is the amount of exposure to pAMP/pBlu plasmid as defined by 10 ml of plasmid.
The...
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