Details about class are:1. In the SYLLABUS2. In the LAB MANUAL3. On the WEBSITE4. In EMAILS

LAB MANUAL:1. Must be carried to every

lab class2. Must be the one for this

quarter3. On the website

Class details

Academic honesty

VERY SERIOUS!1. DO NOT CHEAT

2. DO NOT PLAGIARIZE

3. DO NOT TRY TO GAME THE

SYSTEMOFFENDERS WILL BE TREATED HARSHLY!

1. Automatic “F”

2. Will be reported

3. Notation for the rest of your

academic career

Academic honesty

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

Labs

BE SAFE!

BRING LAB MANUAL

READ LAB MANUAL BEFORE LAB!GENERATE LAB CHECKLIST“To-do” list for experimentsNOT copy/pasted from manual!Hand in before lab; show notebook copy after lab

Lab Safety

BE SAFE!

PAY ATTENTION TO TA INSTRUCTIONS

PAY ATTENTION TO YOUR WORK

WHEN IN DOUBT, ASK!

READ LAB SAFETY/CLEAN UP SECTION

Lab safety

NO FRIENDS/FAMILY

PANTS

CLOSED-TOE SHOES

LAB COATS

GLOVES

WILL BE ASKED TO

LEAVE LAB!

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”

Expectations: You

Self-motivated learning; Independent learningNot just memorization; UnderstandApply 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

Communication

Please participate in surveys and evaluationsCan’t fix what I don’t know is wrong

Give me feedback throughout class (on DB)!

S**t will happen, mostly fixable!

Expectations: Me

Help you learn & understand(NOT “know” or memorize)

Challenge you, make you a little uncomfortableAvailable, approachable

Try to teach you to think scientifically;help you teach yourself

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

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 examsManage time during exams

Class structure

Cytochrome P450

Substrate-H

O2

SubstrateHydroxylase

Class structure

TransformationExpression

ActivityInhibitors

Purification

P450 DNA

Bacteria

Protein

Class structure

Lysozyme Crystallization

Lab 1: Plasmids – why do we need ‘em?

Plasmid – needs to have?

Just “transform” the P450 gene into bacteria?

Lab 1: Plasmids as “expression systems”

Considerations for gene promoter?

Plasmid ori + selection + gene?

Lab 1: Plasmids how to get ‘em?

Difference between plasmid and chromosome?

Major classes of biomolecules?

Separate plasmid and chromosome?

Lab 1: Plasmid prep - overview

Lab 1: Plasmid prep - overview

Lab 1: Plasmid prep - overview

Lab 1: Plasmid prep - overview

Lab 1: Restriction enzymes

Uses?

What is a RE?

Lab 1: Restriction enzyme maps

RE1: 3,2 Kb

Uncut: 5 Kb RE2: 1,4 Kb

RE1+2: 0.5Kb, 1Kb, 3Kb

RE1

RE1

3Kb

2Kb

RE2

RE2

4Kb

1Kb

Lab 1: Restriction enzyme maps

RE1: 3,2 Kb

Uncut: 5 Kb RE2: 1,4 Kb

RE1+2: 0.5Kb, 1Kb, 3Kb

RE1

RE1

RE2

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) & plasmidPredicted fragments with RE1 and RE2?

Think about total size, orientation, change in RE sites

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?

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 sizesRE Map should be presentable!

Lab 1: Theory (from manual)

Expression & regulationof P450 expression in our cells

Design & Advantagesof the T7 expression system

General theoryof protein and plasmid isolation from bacteria