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Lab_6A_P_Green - by Dr. Maria Rapoza and Dr. Helen Kreuzer...

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by Dr. Maria Rapoza and Dr. Helen KreuzerWe would like to acknowledge the contributions ofDr.Doris Helms and Ms. Bobbie Hinson to the ProcedureandDataand Analysis sections.The procedures used in this manual were developed in cooperation with the Dolan DNALearning Center of Cold Spring Harbor Laboratory.BackgroundThe transfer of new DNA into organisms has led to many improvements in our everyday lives.In the biotechnology industry, the transfer of the human genes for insulin and growth hormoneinto bacteria has created bacteria which obligingly produce as much human insulin and humangrowth hormone as we need. Scientists can also take DNA from a deadly organism, divide itinto many pieces, and safely study the individual pieces by introducing the fragments of DNAinto a nonpathogenic host bacterium. These methods have been used to study isolated genesfrom dangerous organisms such as the anthrax bacterium and the AIDS and Ebola viruses.But how is new DNA introduced into an organism? The techniques of gene transfer in higherplants and animals are complex, costly, and extremely difficult even in the researchlaboratory. However, the techniques of gene transfer in E.colibacteria are simple andappropriate for the teaching laboratory.This manual provides detailed information on gene transfer in E.coliincludingbackground information on the history of transformationa discussion of the science of transformationan overview of plasmids readily available for transformation in the teachinglaboratoryand an easy-to-follow procedure for transformation. Discovery oftransformationIn 1928, the English scientist Frederick Griffiths was studying the bacteriumStreptococcuspneumoniae.This organism causes pneumonia, which in 1928 was the leading cause of death inthe Western Hemisphere. Griffiths was working with two strains of S.pneumoniae:one whichcaused disease (a pathogenic strain) and one which did not. The pathogenic form of theorganism produced an external polysaccharide coating that caused colonies of this straingrowing on agar medium to appear smooth. The nonpathogenic strain did not produce thecoating, and its colonies appeared rough. We now know that the polysaccharide coating madethe smooth strain pathogenic by allowing it toescape being killed by the host's immune system.Griffiths' experiments involved injecting mice with theS. pneumoniaestrains. When he used thesmooth strain, the mice became ill and died. When he used the rough strain, theystayedhealthy. Inone series of experiments, Griffiths mixed heat-killed smooth cells (which had noTeacher’s Manual - 1
effect when injected into mice) with living rough cells (which also had no effectwheninjected into mice) and injected the combination into mice. To his surprise, themicebecame ill and died, as if they had been injected with living smooth cells. When GriffithsisolatedS. pneumoniaefrom the dead mice, he found that they produced smooth colonies.

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Term
Spring
Professor
f
Tags
Bacteria, DNA, plasmid dna

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