Exoeriment 3 - Experiment III: Transposon Mutagenesis and...

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Experiment III: Transposon Mutagenesis and Plasmid Inheritance Mark Gaynor MMG 408: Advanced Microbiology Lab November 20 2007
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Introduction Bacterial cells contain chromosomes as their genetic material, but may also contain foreign DNA that the cell has acquired from the environment. Both plasmids and transposons are capable of incorporating into bacterial chromosomes many times leading to genetic mutations in the host DNA. Experimentally, transposons and plasmids also have a variety of purposes that can be utilized in the laboratory. Since bacterial cells must be able to replicate and metabolize on its own, plasmids normally do not carry genes that the cell needs, but instead carry genes that allow a cell to evolve with its surroundings. Since plasmids often confer a measurable phenotype, such as antibiotic resistance, the efficiency at which they transfer into genes can be easily measured. Transposons can have both positive and negative effects that can be observed in the lab; if they insert themselves into a gene that is necessary for normal cellular survival, insertion can be fatal and the cells will die. If the transposon carries a gene for antibiotic resistance it can be life-saving and live in a medium wild-type cells can not. If the transposon does become integrated into a plasmid, we can map its location by cutting it with restriction enzymes and observing their fragment movement on an electrophoresis gel. In this experiment we will be utilizing the plasmid pAT153, which is ampicillin and tetracycline resistant, to test for the activity of the selected Tn5 transposon. The transposon will be introduced through a mutant lambda phage suicide vector. Unable to replicate because of nonsense mutations in their genes, these vectors cannot integrate into the chromosomes, making one less variable to look for in examining the mutated plasma sequences. The suicide vector only serves to carry the Tn5 transposon, and is digested by
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the cell as soon as the transposon leaves. In order to detect the presence or absence of transposon integration, we will use transposons that confer antibiotic resistance, one of the easiest phenotypes to test for in the lab. Depending on whether or not the cells are able to form colonies on selective plates serves as the model for being able to easily tell if a new genetic element has been integrated. We can also accurately predict where integration may have taken place. In addition to experimentally testing for phenotypes, restriction enzymes and gel electrophoresis are tools that help map genetic sequences. In this experiment we will be using HindIII and Pst1 enzymes on purified plasmids with known restriction sites to help detect where the transposon has inserted itself. Materials and Methods
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Exoeriment 3 - Experiment III: Transposon Mutagenesis and...

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