Week 05 - Comp Cells & Transf

Week 05 - Comp Cells & Transf - BIOL 519 Week # 5 -...

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BIOL 519 1 BACKGROUND: This week you will first prepare competent cells of E. coli , and then you will transform the ligation reactions you performed last week into these competent cells. In the transformation process, each of the transformed bacterial cells will take up one circular DNA molecule (plasmid). In this way, we can analyze the phenotype that the individual plasmids confer on the bacteria, and find the plasmids that have the characteristics expected for the desired clones (in this case they will be: Ampicillin resistant, Chloramphenicol resistant, and white on X-gal). Since each transformed bacterial cell will divide to produce a colony, and each cell in the colony will carry an identical copy of the plasmid, the transformation process also provides a method to produce many copies (enough to analyze) of each of the plasmids in the ligation mixture. Potential clones of the desired type will be initially chosen based on the phenotype they confer upon the bacteria on plates. Next week you will isolate the plasmid DNA from promising clones so that we can analyze it in more detail (at the level of the genotype). Transformation - Transformation is a form of genetic exchange in which free DNA is taken up by a bacterial cell and stably maintained. Some bacteria (ie - Streptococcus pneumoniae, Bacillus subtilis, and Hemophilis influenzea ) are capable of taking up free DNA from the environment naturally - Natural Transformation. This is not true of E.coli . Transformation of E.coli requires that the cells be first chemically treated to make them competent – Chemically- Induced Transformation. However, since we know so much about E. coli , and so many useful strains have been developed, E. coli is by far the organism of choice for performing plasmid transformations. Unlike natural transformation systems, E. coli can only be transformed by circular DNA molecules that carry an origin of replication. In other words, the DNA that is taken up by E. coli must be able to replicate independently of the bacterial chromosome to be maintained in the cell. E. coli can take up linear DNA, however, linear DNA will be degraded in the cell and the nucleotides used to make more E. coli DNA - this process is called “eating” not transformation. Competent Cells of E. coli - We understand an enormous amount about the bacterium E. coli . However, the mechanism by which this bacterium becomes “competent” (able to take up DNA from solution) for transformation, and the mechanism it subsequently uses to take up plasmid DNA from its environment (transformation) are only beginning to be understood. In general, E. coli cells are made competent by harvesting cells in early logarithmic growth, then keeping the cells very cold, and treating them with divalent cations, usually including calcium chloride. In our experiment, we will treat the cells with FSB (frozen storage buffer) which contains Calcium Chloride as well as Potassium Chloride, Potassium Acetate and Glycerol. The two potassium
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This note was uploaded on 01/22/2011 for the course BIO 519 taught by Professor Ega during the Spring '10 term at Kansas.

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Week 05 - Comp Cells & Transf - BIOL 519 Week # 5 -...

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