1. Welcome to BISC 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

3. 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)

4. 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

5. Lab Grading Series 1 –Homework assignments (3)35 –Lab report 40 Series 2 –Homework assignment (1)15 –Lab report 45 Series 3 –Group Presentation25 –Partial Lab report35 “P” points—Participation & Preparation 5 TOTAL: 200

6. 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

7.  -galactosidase: our enzyme of choice –Our  -gal is the Escherichia coli (E. coli) version Lactose Glucose+ Galactose Beta-galactosidase

8. How is  -gal expression normally regulated? lac operon

9. 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:

10. 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-  -gal. (The 6xHis tag will be used in the purification process.)

11. 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)

12. 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)

13. 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)

14. The Four Levels of Protein Structure

15. Interactions that contribute to tertiary & quaternary structure

16. X-ray crystallography as a means for determining a protein’s structure at the atomic level

17. 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.