1. Details about class are: 1. In the SYLLABUS 2. In the LAB MANUAL 3. On the WEBSITE 4. In EMAILS LAB MANUAL: 1. Must be carried to every lab class 2. Must be the one for this quarter 3. On the website Class details

2. Academic honesty VERY SERIOUS! 1. DO NOT CHEAT 2. DO NOT PLAGIARIZE 3. DO NOT TRY TO GAME THE SYSTEM OFFENDERS WILL BE TREATED HARSHLY! 1. Automatic “F” 2. Will be reported 3. Notation for the rest of your academic career

3. Academic honesty Signed copy of academic honesty statement -Upload on Dropbox -Do it today!!!

4. Labs BE SAFE! BRING LAB MANUAL READ LAB MANUAL BEFORE LAB! GENERATE LAB CHECKLIST “To-do” list for experiments NOT copy/pasted from manual! Hand in before lab; show notebook copy after lab

5. Lab Safety BE SAFE! PAY ATTENTION TO TA INSTRUCTIONS PAY ATTENTION TO YOUR WORK WHEN IN DOUBT, ASK! READ LAB SAFETY/CLEAN UP SECTION

6. Lab safety NO FRIENDS/FAMILY PANTS CLOSED-TOE SHOES LAB COATS GLOVES

7. Class goals 1. Experimental techniques “Doing” 2. Critical thinking, experimental design “Thinking,” “Independence” 3. Reading & writing (papers, lab reports, etc.) “Communication” 4. Discipline & Rigor “Self-management”

8. Expectations: You Self-motivated learning; Independent learning Not just memorization; Understand Apply yourself, ask yourself questions ( What if? How would I? ) Be a good lab citizen; Be safe Active participant in class Be a little uncomfortable Communicate – surveys, evals, DB

9. Communication Please participate in surveys and evaluations Can’t fix what I don’t know is wrong Give me feedback throughout class (on DB)! S**t will happen, mostly fixable!

10. Expectations: Me Help you learn & understand (NOT “know” or memorize) Challenge you, make you a little uncomfortable Available, approachable Try to teach you to think scientifically; help you teach yourself

11. Dr. K Set up lunch/coffee meetings! Sarchasm! Please don’t take it personally! Role = Guide + Mentor (use me!) Go to office hours! Current research: How to improve learning

12. Exams/Quizzes Need to think, not just memorize Study material – develop working memory of concepts Open book = Harder questions Learn to apply concepts to do well in exams Manage time during exams

13. Class structure Cytochrome P 450 Substrate-H O2O2 Substrate Hydroxylase

14. Class structure Transformation Expression Activity Inhibitors Purification P 450 DNA Bacteria Protein

15. Class structure LysozymeCrystallization

16. Lab 1: Plasmids – why do we need ‘em? Plasmid – needs to have? Just “transform” the P 450 gene into bacteria?

17. Lab 1: Plasmids as “expression systems” Considerations for gene promoter? Plasmid ori + selection + gene?

18. Lab 1: Plasmids how to get ‘em? Difference between plasmid and chromosome? Major classes of biomolecules? Separate plasmid and chromosome?

19. Lab 1: Plasmid prep - overview

23. Lab 1: Restriction enzymes Uses? What is a RE?

24. Lab 1: Restriction enzyme maps RE1: 3,2 Kb Uncut: 5 KbRE2: 1,4 Kb RE1+2: 0.5Kb, 1Kb, 3Kb RE1 3Kb 2Kb RE2 4Kb 1Kb

25. Lab 1: Restriction enzyme maps RE1: 3,2 Kb Uncut: 5 KbRE2: 1,4 Kb RE1+2: 0.5Kb, 1Kb, 3Kb RE1 RE2

26. Lab 1: Restriction enzyme cloning Digest plasmid with RE1 ( incomplete ) Previous plasmid Digest gene of interest (4Kb) with RE1 “Ligate” gene (no RE1/2 sites) & plasmid Predicted fragments with RE1 and RE2? Think about total size, orientation, change in RE sites

27. Lab 1: Restriction enzyme maps RE1: 3,2 Kb Uncut: 5 Kb RE2: 1,4 Kb RE1+2: 0.5Kb, 1Kb, 4Kb RE1: 4,3,2 Kb Uncut: 9 Kb RE2: 1,8 Kb RE1+2: 0.5Kb, 1Kb, 7Kb Conclusion?

28. Lab 1: Plasmid map assignment TABLE (apprx.) fragment sizes DUE Week 4 RE Map (with relative orientation of sites) RE Map does not require exact fragment sizes RE Map should be presentable!

29. Lab 1: Theory (from manual) Expression & regulation of P 450 expression in our cells Design & Advantages of the T7 expression system General theory of protein and plasmid isolation from bacteria