Welcome to BISC 220 Cell Physiology Lab

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Welcome toBISC 220 Cell Physiology Lab

• Lab Instructor Jennifer
• Hood-DeGrenier
• Office SC 376A, x3313
• Research Lab SC 311, x3387
• Email jhooddeg_at_wellesley.edu
• Office Hours
• Tues. 130-230 pm
• Thurs. 930-1030 am
• Or e-mail to schedule an appointment

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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)

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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

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Lab Grading

• Series 1
• Homework assignments (3) 35
• Lab report 40
• Series 2
• Homework assignment (1) 15
• Lab report 45
• Series 3
• Group Presentation 25
• Partial Lab report 35
• P pointsParticipation Preparation 5
• TOTAL 200

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Lab 1

• Induction of b-galactosidase (b- gal) expression
  in E. coli
• RasMol
• Investigation of the structure of ?-gal
• ClustalW
• Identification of amino acid residues conserved
  among b-gal proteins from different species

Next week purification of b-gal for study of
its enzymatic properties
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b-galactosidase our enzyme of choice
Lactose Glucose Galactose
Beta-galactosidase

• Our b-gal is the Escherichia coli (E. coli)
  version

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How is b-gal expression normally regulated?
lac operon
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I. E. coli BL21(DE3) genetically engineered to
express T7 RNA polymerase in the presence of IPTG
Our system how to make lots of b-gal!
A two-part process
IPTG binds lac repressor, prevents it from
interfering with the lac promoter and turns on T7
Pol expression
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II. pET-14 plasmid in this E. coli strain
T7 promoter
lacZ encodes ?-gal with 6 histidines (His) added
as a tail (affinity tag)
6xHis
Expression of T7 polymerase causes expression of
large amounts of 6xHis-b-gal. (The 6xHis tag will
be used in the purification process.)
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ProtocolThings 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 sinkput 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)

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While your bacteria are making lots of
6xHis-?-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)

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A quick review of protein structure

• Levels of structure primary, secondary, tertiary
  quaternary
• Secondary structure elements a-helices
  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)

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The Four Levels of Protein Structure
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Interactions that contribute to tertiary
quaternary structure
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X-ray crystallography as a means for determining
a proteins structure at the atomic level
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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.