Transcript Welcome to BISC 220 Cell Physiology Lab

Welcome to
BISC 220
Cell Physiology Lab
Lab Instructor: Jennifer
Hood-DeGrenier
Office: SC 376A, x3313
Research Lab: SC 311, x3387
Email: [email protected]
Office Hours:
Tues. 1:30-2:30 pm
Thurs. 9:30-10:30 am
Or e-mail to schedule an appointment
The Four Strands of Modern
Cell Biology
• Cytology: observation of cells by
microscopy
• Biochemistry: reductionist approach;
in vitro study of biological
molecules
• Genetics: study of the effect of
heritable information (DNA) on cell
behavior/attributes; use of mutants
to study cellular processes
• Bioinformatics: application of
computer algorithms to the analysis
of large databases of biological
information (genomics/proteomics)
BISC 220 Lab Overview
• Series1 (Biochemistry)
– Protein purification & enzyme kinetics using the
enzyme b-galactosidase
• Molecular modeling & database search
• Recombinant protein induction & purification
by affinity chromatography
• Quantitative & qualitative assessment of
purification success (gel electrophoresis)
• Quantitative enzyme kinetics assays,
including determination of the effect of an
inhibitor
• Series 2 (Genetics)
– Analysis of the secretory pathway in budding
yeast
• Genetic assay to identify/characterize mutants
defective in secretion
• Western Blot to assess location of secretion
defect
• Series 3 (Cytology)
– Tissue culture & the cytoskeleton
• Learning cell culture techniques
• Determining the effect of varying
concentrations of a drug on the actin
cytoskeleton & cell viability by fluorescence
microscopy & flow cytometry
Lab Grading
• Series 1
– Homework assignments (3)
– Lab report
35
40
• Series 2
– Homework assignment (1)
– Lab report
15
45
• Series 3
– Group Presentation
– Partial Lab report
25
35
• “P” points—Participation &
Preparation
5
TOTAL: 200
Lab 1
• Induction of b-galactosidase (bgal) expression in E. coli
• RasMol
– Investigation of the structure of
b-gal
• ClustalW
– Identification of amino acid
residues conserved among bgal proteins from different
species
Next week: purification of b-gal for
study of its enzymatic properties
b-galactosidase: our
enzyme of choice
Lactose
Glucose+ Galactose
Beta-galactosidase
– Our b-gal is the Escherichia
coli (E. coli) version
How is b-gal expression
normally regulated?
lac operon
Our system: how to make lots of b-gal!
A two-part process:
I. E. coli BL21(DE3): genetically
engineered to express T7 RNA
polymerase in the presence of IPTG
lac promoter
lacO
T7 polymerase
lac repressor
IPTG
(resembles
natural
inducer of
lac operon)
IPTG binds lac repressor, prevents it
from interfering with the lac promoter
and turns on T7 Pol expression
II. pET-14 = plasmid in
this E. coli strain
T7 promoter
lacZ
6xHis
encodes b-gal with 6 histidines
(His) added as a tail (affinity
tag)
Expression of T7 polymerase
causes expression of large
amounts of 6xHis-b-gal. (The 6xHis
tag will be used in the purification
process.)
Protocol:
Things to Remember
• Think about aseptic technique (avoid
contaminating your culture!)
• Make flow chart of procedure and
record all results in lab notebook
• Do not discard anything contaminated
with bacteria in sink—put growth
medium in waste container or back in
flask (must be treated with bleach)
• Give labeled cell pellets to instructor
for freezing:
– Pre-IPTG induction (small aliquot in tube)
– After IPTG induction (small aliquot in
tube)
– After IPTG (remainder in centrifuge
bottle)
While your bacteria are
making lots of 6xHis-b-gal…
• Calibrate micropipets
• Look at CD animation of pdb
file
• Follow RasMol tutorial in
Appendix 1 Lab 1 (groups of 2)
• Follow ClustalW instructions in
Appendix 2 Lab 1 (same
groups as RasMol)
A quick review of
protein structure
• Levels of structure: primary,
secondary, tertiary & quaternary
• Secondary structure elements: ahelices & b-sheets
• R-group interactions
– Salt bridges (ionic interactions),
Hydrogen bonds, van der Waals
forces, hydrophobic interactions,
disulfide bonds
• Use of X-ray crystallography to
“solve” protein structures (important
for determining enzyme
mechanisms, designing drugs,
engineering mutations that alter
protein function)
The Four Levels of
Protein Structure
Interactions that contribute to
tertiary & quaternary structure
X-ray crystallography as a means for
determining a protein’s structure at the
atomic level
Homework
• Do individually
• Due next lab; 10 points
• Create a figure with a correctly
formatted legend from your
saved RasMol picture of the
active site of b-gal.
• Include a paragraph (up to 1
page) describing what you
learned about the active site. Try
to relate the ClustalW analysis to
the structural analysis.
• May consult references listed at
end of HW assignment in lab
manual.