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Expression of the lacZ Gene from the ibpB Heat Shock Protein

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Expression of the lacZ Gene from the ibpB Heat Shock Protein Ebee Hornbeck & Sheena Donald The goal of our project is to successfully create a plasmid containing the ... – PowerPoint PPT presentation

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Title: Expression of the lacZ Gene from the ibpB Heat Shock Protein


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
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 ß-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 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
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