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Expression of the lacZ Gene from the ibpB Heat Shock Protein

Expression of the lacZ Gene from the ibpB Heat Shock Protein. Ebee Hornbeck & Sheena Donald.

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Expression of the lacZ Gene from the ibpB Heat Shock Protein

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  1. Expression of the lacZ Genefrom the ibpB Heat Shock Protein Ebee Hornbeck & Sheena Donald

  2. 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

  3. 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. Escherichia coli

  4. 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

  5. The functions of the hsps were unknown at first, but now they are thought to regulate and assist with protein folding within the cell

  6. The lac Operon

  7. 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

  8. 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

  9. 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

  10. 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 • September 27 – Gel electrophoresis to verify correct PCR product • October 1 – Transform plasmid in E. Coli • October 4 – Screen E. Coli for plasmid • October 8 – Use spectrometer to measure level of transcription at 30C • November 1 – Have completed spectrometer readings to measure level of transcription at 20C, 37C and 45C. • November 22 – Have results and report completed.

  11. Budget

  12. References • Gross, Carol A. Function and Regulation of the Heat Shock Proteins • Griffiths, A.J.F., Gelbart, W., Lewontin, R.C., Miller, J.H. Modern Genetic Analysis. New York, 2002. • Liang, Sung-Tzu, Dennis, Patrick, Bremer, Hans. Expression of lacZ from the Promoter of Escherichia coli spcOperon Cloned into Vectors Carrying the W205 trp-lac Fusion. Journal of Bacteriology, December 1998, p.6090- 6100. • Watson, James D. et al. Molecular Biology of the Gene. San Francisco, 2004.

  13. Grade Agreement

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