Welcome toBISC 220

Cell Physiology Lab

Lab Instructor: Jennifer

Hood-DeGrenier

Office: SC 376A, x3313

Research Lab: SC 311, x3387

Email: jhooddeg@wellesley.edu

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 -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) 35– Lab report 40

• Series 2– Homework assignment (1)

15– Lab report 45

• Series 3– Group Presentation

25– Partial Lab report 35

• “P” points—Participation & Preparation 5

TOTAL: 200

Lab 1

• Induction of -galactosidase (- gal) expression in E. coli

• RasMol

– Investigation of the structure of -gal

• ClustalW

– Identification of amino acid residues conserved among -gal proteins from different species

Next week: purification of -gal for study of its enzymatic properties

-galactosidase: our enzyme of choice

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

Lactose Glucose+ GalactoseBeta-galactosidase

How is -gal expression normally regulated?

lac operon

I. E. coli BL21(DE3): genetically engineered to express T7 RNA polymerase in the presence of IPTG

IPTG binds lac repressor, prevents it from interfering with the lac promoter and turns on T7 Pol expression

lacO T7 polymerase

lac promoter

lac repressor

IPTG(resembles natural inducer of lac operon)

Our system: how to make lots of -gal!

A two-part process:

II. pET-14 = plasmid in this E. coli strain

T7 promoter

lacZencodes -gal with 6 histidines (His) added as a tail (affinity tag)

6xHis

Expression of T7 polymerase causes expression of large amounts of 6xHis--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--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: -helices & -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 -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.