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 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 functions of the hsps were
unknown at first, but now they are
thought to regulate and assist with
protein folding within the cell
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
• 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.
Budget
Plasmid containing lacZ,
restriction enzymes and
antibiotic resistance gene
~$350.00 (20 μg)
BD Bioscience
Or
Invivogen
PCR Kit
Primer
Supplies for gel
electrophoresis
Two restriction enzymes
X-gal and IBPT solutions
Agar plates containing
appropriate antibiotic
~$0.69 per nucleotide
Supplied by UE
Biology
~$106.00
New England Biolabs
~$57.00 per gram
Gold BioTechnology
~$85.00
Invivogen
References
• Gross, Carol A. Function and Regulation of the Heat Shock
Proteins
• Griffiths, A.J.F., Gelbart, W., Lewontin,
R.C., Miller,
Modern Genetic Analysis. New York, 2002.
J.H.
• 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.
Grade Agreement
Finding correct promoter sequence,
determining necessary restriction
enzymes and nucleotide sequence of
primer
5
PCR of promoter
5
Gel electrophoresis for verification
5
Insert promoter in plasmid
5
Insert plasmid in E. Coli
5
Screen for plasmid in E.Coli
5
Screen for β-galactosidase at 30°C
5
Screen for β-galactosidase at 20°C
5
Screen for β-galactosidase at 37°C
5
Screen for β-galactosidase at 45°C
5