Appendix B

NORTH CAROLINA AGRICULTURAL AND TECHNICAL STATE UNIVERSITY Course Syllabus

Course Information Course Number & Section Course Title Term Location Days & Times Website Biology 642 Section 001 (CRN 17632) Designer Proteins and Society Fall 2012 224 Barnes Hall Tuesday & Thursday 12:30pm - 1:45pm http://blackboard.ncat.edu

Professor Contact Information Professor Dr. Justin Shaffer Email Address jfshaffe@ncat.edu Office Phone 724-301-2712 Office Location G10 Barnes Hall Office Hours Tuesday 10:00am 12:00pm Thursday 2:00pm 4:00pm Course Description and Goals Proteins can be so much more than what you find in a burger or a nutritional supplement! Did you know that scientists and engineers can design and produce custom proteins to meet specific functional and technical needs? Whether they are for medical, pharmaceutical, industrial, agricultural, or environmental settings, recombinant proteins are used in a variety of ways to benefit society. This course provides an introduction to the fascinating and diverse field of recombinant proteins. Once we cover the basics of how recombinant proteins are made, youll learn how to use recombinant DNA technology to design protein expression vectors. Next, youll learn how various expression systems (bacterial, insect, and animal cells) and purification schemes (chromatography, centrifugation, dialysis, etc) are used to produce and purify recombinant proteins. Youll also have the opportunity to perform a realistic laboratory research project to express, purify, and functionally assess a recombinant protein. Throughout the course well discuss the many ways that recombinant proteins are used in modern day medicine, industry, and agriculture, and will discuss their societal and ethical impacts. Specifically, at the end of this course, you will be able to Define and explain key fundamental terms and concepts of protein biochemistry Apply molecular biology methods and recombinant DNA technology to create recombinant protein expression vectors Design an expression and purification scheme for a recombinant protein based on the proteins biochemical properties Develop a design proposal to clone, express, purify, and assess a recombinant protein Explain how recombinant proteins are used in medical, pharmaceutical, industrial, environmental, and agricultural applications and why they are so important to society Explain and evaluate the ethical implications surrounding recombinant DNA technology and the use of recombinant proteins Design, execute, and troubleshoot laboratory experiments and methods relating to recombinant protein expression and purification

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Page 2 More specific learning objectives and goals are presented for each unit as described in the course schedule below, and will also be provided at the beginning of each day of class. Prerequisites Molecular biology (BIOL 401). If you do not meet the prerequisites please contact me. Required Textbooks and Materials There are no required textbooks for this course. You might want to consult your biology, molecular biology, or biochemistry textbooks from time to time to help brush up on some material. Reading materials will be posted on Blackboard for you to read and print out. Course Requirements and Evaluation Active Learning: You might be used to taking courses in which the professor simply lectures the entire class period. This course is going to be very different, as it is going to be an active classroom. By active I mean that you will be required to interact with myself and your fellow students to learn the material presented in this course. We will be using activities such as small group work, case studies, and class discussions to actively engage in the learning process. In order to have an active classroom, attendance is extremely important. By attending class and working through these active learning exercises you will develop critical thinking and problem solving skills that are essential to performing well in this course and others. Recombinant Protein of the Day: Recombinant proteins are essential to medicine, industry, agriculture, and the environment. To highlight the importance of recombinant proteins in our society, each day of class will feature a Recombinant Protein of the Day. During these short, 5 minute presentations, we will learn about the function of the recombinant protein, how it is used by society, and how it is produced. I will give the first handful of presentations, but then you as a class will be responsible for presenting the remainder. The goal is not to learn everything possible about the specific recombinant protein, but rather to be exposed to the many, many ways that recombinant proteins positively affect our society! See the handout for more details. Design Project: The overall goal of this course is that you will be able to design and map out a procedure to clone, express, purify, and functionally assess a recombinant protein. This project will be in the form of a written proposal. The proposal will include the following sections: 1) background on the biological importance of the protein you want to produce; 2) design of the cloning process (choice of vector, primer design, choice of restriction enzymes, etc); 3) choice of the expression system; 4) design of a purification scheme (type of chromatography, etc); and 5) design of experiments to assess the function of the protein. All sections will require justifications as to why you designed the process the way you did. Throughout the semester, there will be checkpoints where you will have to turn in parts of the project to keep you on track. The final project will be due at the end of the semester. Further instructions and guidelines for the project will be given out in class. Laboratory Project: During the last few weeks of this course you will have the chance to take part in a realistic research project where you will express, purify, and functionally assess a recombinant protein, dihydrofolate reductase (DHFR), an important enzyme involved in the synthesis of nucleic acid precursors. We will be using the Bio-rad Biotechnology Explorer Protein Expression and Purification series of modules that will give you the opportunity to grow E. coli bacteria to express DHFR, to purify DHFR using affinity chromatography, and to assess the enzymatic activity of DHFR using a spectrophotometric assay. Assessments of the lab portion of the class will include pre-lab assignments, in-class worksheets, and a written lab report. Further instructions and guidelines will be given out in class.

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Page 3 Guest Speakers and Field Trip: In this course you will be exposed to a variety of techniques and principles that are used in the biotechnology sector. To give you more insight into this type of industry, we will be having a guest speaker in class from a local biotechnology company to share their experiences in working with recombinant proteins in industry. We will also be taking a field trip to a local biotechnology company to see the production of recombinant proteins on a large scale. Further details will be announced in class. Evaluation: There will be multiple grading opportunities in this course, giving you many chances to do well. A summary of the various grading opportunities is given below. Pre-Test: The pre-test will cover basic DNA and protein concepts that you need to know to do well in this course. The pre-test is worth 50 points, or 5% of your course grade. Recombinant Protein of the Day: You are required to give a 5 minute presentation describing the recombinant protein of your choice. The due date is variable, as you will sign up for a specific date to present. This presentation is worth 50 points, or 5% of your course grade. Quizzes: Throughout the semester, we will have brief, unannounced in-class quizzes that will assess your knowledge of the course material. If you miss a quiz, you will not be able to make it up. Quizzes are worth 50 points, or 5% of your course grade. Homework Assignments: There will be four homework assignments that will help you stay up to date on the course material. Homework assignments will be due at the beginning of class on the day they are due. Homework due dates are listed in the detailed course schedule below. Homework assignments are worth 100 points, or 10% of your course grade. Exams: There will be two take-home exams in this course, with each worth 10% of your course grade. The first exam will be due on September 25th and will cover material from Units 1 and 2. The second exam will be due on October 23rd and will cover material from Unit 3. Laboratory Project: There will be several components of the lab project, including pre-lab assignments, in-class worksheets, and a final written report (due December 5th at 5pm). All of these activities will add up to 250 points, or 25% of your course grade. More details about the laboratory project will be handed out in class. Design Project: There will be several due dates throughout the semester where you will have to turn in updates on your design project. The project will culminate in oral presentations held during the final exam period (Friday December 7 from 1 3pm). All of the components of the design project will add up to 300 points, or 30% of your course grade. More details about the design project will be handed out in class. The breakdown for course points is as follows: Pre-Test Protein of the Day Quizzes Homework Exams Laboratory Project Design Project Total 5% 5% 5% 10% 20% 25% 30% 100% 50 points 50 points 50 points 100 points 200 points 250 points 300 points 1000 points

(see handout for details) (4 at 25 points each) (2 at 100 points each) (see handout for details) (see handout for details)

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Page 4 Based on the above point structure, you can calculate your grade at any time during the semester (ask for help if you need it), and you should calculate your grade regularly to keep track of how you are doing in the course. The number of points will be converted to letter grades based on the following: 895 1000 points 795 894 points 695 794 points 595 694 points Less than 595 points A B C D F

Course Policies Courtesy to Fellow Students: We are going to have a positive learning environment in this class, so courtesy to your fellow students (and to me!) is imperative. Do you want to be distracted while trying to learn? Probably not, so please treat your classmates as you want to be treated. This includes putting your cell phones on silent before class starts, not using cell phones during class, limiting side conversations and comings and goings during class, not reading newspapers or doing the crossword puzzles, and other possible distractions. Attendance: Attendance is vital to succeeding in this course. Participation in the active learning activities during class will help you develop your critical thinking and problem solving skills which will help you on the exams in this course and in other courses. Finally, attendance is required by North Carolina A&T State University, and failure to attend class regularly will result in a reduction of your grade and possible failure of the course. Academic Integrity: Enrollment in this class means that you agree to abide by the expectations of North Carolina A&T State University regarding academic integrity. For specific information, refer to your Student Handbook. Also, refer to the most current Undergraduate Bulletin for the academic dishonesty policy. The Universitys Academic Honor Code will be strictly enforced. Your responsibilities in the area of honor include, but are not limited to, avoidance of cheating, plagiarism and improper or illegal use of technology. Your assignments are expected to be your own work. If you have questions, please ask. Integrity is an important characteristic that should be exemplified in the lives of all North Carolina A & T State University students. Dishonesty will not be tolerated in any form in this class. Any student caught cheating on an examination or any other class assignment will be given a grade of zero for that examination or class activity and reported to University officials for further disciplinary actions. Plagiarism (i.e. citing information written by another person without referencing the persons work) will also lead to a grade of zero for the assignment. Changing a few words in material taken from a book or the internet without referencing the author of the material is still plagiarism. Late Work: No late work will be accepted. All assignments are due at the beginning of class on the day that they are due. Please plan accordingly to make sure your homeworks and other assignments are turned in on time. Make-up Work: If you have an official university excuse for missing an exam (death in the family, sickness, university activity), then a make-up exam will be possible. Please see me ahead of time if you know you will miss an exam due date. Quizzes cannot be made up. Field Trips: We will be going on a field trip in this course. See the university policy on field trips and class travel. More details will be announced in class.

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Page 5 Course Schedule The following is the schedule for the course. The course is broken up into four units, with the first three units being classroom-based, and the last unit being a hands-on laboratory project. Due dates for homework (HW), exams, design project (DP), and pre-lab (Pre) assignments are listed when appropriate. See design project and laboratory project hand-outs for more details on these assignments. The schedule is subject to change. Unit 1: Introduction to Recombinant Proteins BIG QUESTION: What is a recombinant protein? At the end of this unit you will be able to Evaluate the use of recombinant DNA technology to make recombinant proteins Outline the major steps in the design and production of a recombinant protein Use online tools and databases to research and create recombinant proteins Thursday August 16 Tuesday August 21 Thursday August 23 Tuesday August 28 Course introduction and overview Insulin: the first recombinant drug How to make a recombinant protein Online tools for protein design Unit 2: Recombinant DNA Technology BIG QUESTION: How do you use recombinant DNA technology to make proteins? At the end of this unit you will be able to Outline the steps necessary to prepare a functional expression vector Identify and describe the essential features of cloning and expression vectors Design PCR primers to clone a gene of interest Explain how restriction enzymes are used to clone genes Explain how to screen for positive recombinant clones Thursday August 30 Tuesday September 4 Thursday September 6 Tuesday September 11 Thursday September 13 Tuesday September 18 Overview and RNA and cDNA preparation Polymerase chain reaction & primer design Plasmids and Expression vectors DNA manipulation (restriction enzymes & ligation) Transformation and screening of recombinants Verification of vector contents & DNA sequencing

Pre-test due

HW 1 DP 1 HW 2

Unit 3: Recombinant Protein Expression and Purification BIG QUESTION: How do you produce and purify a recombinant protein? At the end of this unit you will be able to Compare and contrast recombinant protein expression systems Describe protein purification methods and technologies Evaluate purification methods for a given protein given its properties Describe methods used to assess protein purity Explain how to quantify and assess recombinant protein activity/function Thursday September 20 Tuesday September 25 Thursday September 27 Expression systems Cell culture growth, induction, and expression Crude purification methods

Exam 1

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Page 6 Tuesday October 2 Thursday October 4 Tuesday October 9 Thursday October 11 Tuesday October 16 Thursday October 18 Tuesday October 23 Chromatography I Chromatography II Fall Break no class Assessment of protein purity and yield Assessment of protein function Scale-up of protein production schemes Guest lecture X-ray crystallography Unit 4: Laboratory Project BIG QUESTION: How do you express, purify, and functionally assess DHFR? At the end of this unit you will be able to Grow, induce, and handle E.coli bacteria using sterile techniques Express and purify DHFR from E.coli using affinity chromatography Assess the purity and function of DHFR using SDS-PAGE and enzymatic assays Collect and analyze data in a realistic research project Thursday October 25 Tuesday October 30 Thursday November 1 Tuesday November 6 Thursday November 8 Tuesday November 13 Thursday November 15 Tuesday November 20 Thursday November 22 Tuesday November 27 Thursday November 29 Introduction to project & basic lab techniques Lyse cells Crude purification of DHFR Affinity purification of DHFR Desalting and SDS-PAGE Concentration measurement of DHFR Enzymatic assay of DHFR activity Data analysis Thanksgiving break no class Guest speaker Course summary and evaluations Pre 1 Pre 2 Pre 3 Pre 4 DP 3, Pre 5 Pre 6 Pre 7 HW 3

DP 2 HW 4 Exam 2

DP 4

Final lab report due December 5 at 5pm Final exam period: Friday December 7 from 1 3pm

DP 5