1. Expression of the lacZ Gene from 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. 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.

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 Plasmid containing lacZ, restriction enzymes and antibiotic resistance gene ~$350.00 (20 μg)BD Bioscience Or Invivogen PCR Kit Primer~$0.69 per nucleotide Supplies for gel electrophoresis Supplied by UE Biology Two restriction enzymes~$106.00New England Biolabs X-gal and IBPT solutions~$57.00 per gramGold BioTechnology Agar plates containing appropriate antibiotic ~$85.00Invivogen

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 spc Operon 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 Finding correct promoter sequence, determining necessary restriction enzymes and nucleotide sequence of primer 5 PCR of promoter5 Gel electrophoresis for verification5 Insert promoter in plasmid5 Insert plasmid in E. Coli5 Screen for plasmid in E.Coli5 Screen for β-galactosidase at 30°C5 Screen for β-galactosidase at 20°C5 Screen for β-galactosidase at 37°C5 Screen for β-galactosidase at 45°C5