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Gene regulation and expression
6
Gene regulators are required to switch genes on and off to prevent from wasting vital cell resources when they are not needed. In prokaryotic cells, there are two levels of gene regulation: transcriptional and translational. Transcriptional controls regulate mRNA production, and translational controls regulate the production of a particular protein.
Operons are portions of cells that make proteins for the cell. Operons either regulate structural genes, which code for the proteins required for normal function of the cell, or regulator genes that code for proteins used by other genes.
The arabinose (ara) operon uses both negative and positive control. Three different genes are expressed in this operon: araA, araB and araD. These genes convert enzymes within the operon to its regular form.
A fourth gene, araC, makes proteins that regulate structural genes. AraC is required for the transcription of the other three genes, and can prevent it's own transcription to regulate araC levels. When araC levels are low, more araC will be produced in order to help sustain the cell. The araC must bind to particular sites on the operon to begin transcription of the other three genes. When arabinose is present, it binds to araC, creating a new shape, and promotes transcription by the attachment of RNA polymerase to DNA. Arabinose is therefore required for GFP expression, in this experiment.
Ampicillin is an antibiotic, part of the penicillin drug family. It fights bacteria within the body, and can kill gram-negative or gram-positive bacteria. Ampicillin contains an amino group which allows it to reach gram-negative bacteria.
Ampicillin works by becoming present in the final stage of bacteria synthesizing into the cell wall, through binary fission. This drug has been used since 1961 to treat bacterial infections such as ear and bladder infections, gonorrhea, and E.coli infections.
It is expected that by using heat shock for bacterial transformation, the pGLO plasmid will be inserted into an E.coli bacterium, and when cultured on an agar plate present with arabinose, the colonies will glow green under UV light.
It is predicted that the +pGLO LB/amp/ara plate in the controlled experiment will contain many colonies that glow green under UV light.
The prediction for the variable experiment, using the homemade transformation solution, is that there will still be colonies on the +pGLO LB/amp/ara plate that glow under UV light, just not as many as in the control experiment.
The plates with both the plasmid and ampicillin , and the plates with no plasmid and no ampicillin will grow colonies; -pGLO LB/amp will not grow bacteria.
Gene regulation and expression
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