ExpressionofthelacZGenefromtheibpBHeatShockProtein.pptVIP

Expression of the lacZ Genefrom the ibpB Heat Shock Protein Ebee Hornbeck Sheena Donald The goal of our project is to successfully create a plasmid containing the coding sequence for the lacZ gene and a promoter from a heat shock gene, IbpB. We will then insert the plasmid into E. coli cells and observe the transcription rate of the lacZ gene at various temperatures Escherichia coli E. coli can cause foodborne illness. Harmless strains of E. coli can be found widely in nature, including the intestinal tracts of humans and warm-blooded animals. Heat Shock Proteins Heat shock proteins are present in cells under normal conditions, but are expressed at high levels when exposed to a sudden temperature jump or other stress The lac Operon The plasmid must contain restriction enzymes, the lacZ gene and a gene for a certain antibiotic resistance. All E. coli taking up the plasmid with form a colony on a medium with that certain antibiotic. E. coli forming colonies will be treated with X-gal and then be exposed to various temperatures. E.Coli transcribing the lacZ gene will appear blue due to reaction of β-galactosidase and X-gal The rate of transcription of the lacZ gene can be determined by the shade of blue. The darker the E. coli, the higher rate of transcription. A spectrometer will be used to measure the intensity of the blue color Protocol Find promoter sequence of ibpB gene Find a good primer for PCR of ibpB promoter, which will also include sticky ends of restriction sites PCR promoter Use gel electrophoresis to verify PCR product Insert plasmid into E. coli Screen E. coli for plasmid using antibiotic medium and X-gal Expose E. coli containing the plasmid to various temoeratures Use spectrometer to measure level of transcription Timeline September 10 – Order and locate all necessary supplies September 13 – Get promoter sequence and figure out necessary restriction enzymes; Develop and order primer and restriction enzymes. September 24 –PCR Septem