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JOHNS HOPKINS UNIVERSITY Instructor Name: Robert D. Horner, Ph.D. Instructor Contact Information Department of Course Title: Foreign Gene Expression Laboratory 179 UTL, Department of Biology 410-516-8067 or -7281 [email protected] Instructor Office Hours/Location: During lab, Mon-Fri 9 am – 2 pm Class Hours: 9 am- noon; 1 – 2 pm Course Number: 020.296 Distribution: Credits: 2 Dates: Monday – Friday, Jan. 9 – 27, 2017 Classroom: UTL 174 COURSE DESCRIPTION This laboratory will introduce molecular cloning techniques that allow bacteria to be used to produce a particular gene product. Recombinant plasmids, carrying a fusion protein gene, will be constructed and used to transform competent E. coli, and the gene products isolated. Prerequisite: permission of instructor. COURSE LEARNING OBJECTIVES Students should obtain a good working knowledge and basic understanding of the following areas: 1 Structure, isolation and use of bacterial plasmids as vectors for selected genes. 2 Use of restriction enzymes to cleave plasmids to make specific DNA fragments. 3 Isolation and characterization of DNA fragments and proteins by gel electrophoresis. 4 Making recombinant plasmids by ligation; transformation and selection of transformed bacteria. 5 Expression of recombinant genes and purification of the gene products. REQUIRED MATERIALS TEXTBOOKS: Protocol notebook supplied. SUPPLIES: all provided by instructor COURSE WEBSITE: on Blackboard EVALUATION AND GRADING S/U grades will be based on the following assessments: ASSESSMENT TYPE Assignments: Seven homework problem sets Lab report detailing the construction of the recombinant plasmid and the expression and purification of the fusion protein. Exams: none Lab Notebook: a complete, accurate and up to date record of all work done in the lab. PERCENT OF FINAL GRADE 40% 50% 10% ATTENDANCE POLICY Students are required to attend all lab sessions. JOHNS HOPKINS POLICIES AND SUPPORT SERVICES This course is governed by the policies set forth in The Johns Hopkins University Undergraduate Student Handbook, which contains information on a wide variety of topics, such as support services, and policies relating to student rights and responsibilities. This course is governed by the policies set forth in this document. Some JHU student support services you may find useful include: SUPPORT SERVICE LOCATION PHONE NUMBER / WEBSITE Library E-Reserves http://www.library.jhu.edu/services/forms/reserves.html [email protected] Summer & Intersession Programs 3505 N. Charles Street, Suite 101 410-516-4548 http://www.jhu.edu/intersession CLASSROOM ACCOMODATIONS FOR STUDENTS WITH DISABILITIES If you are a student with a documented disability who requires an academic adjustment, auxiliary aid or other similar accommodations, please contact The Office of Student Disability Services at [email protected], call 410-516-4720 or visit 385 Garland Hall. STATEMENT OF DIVERSITY AND INCLUSION Johns Hopkins University is a community committed to sharing values of diversity and inclusion in order to achieve and sustain excellence. We believe excellence is best promoted by being a diverse group of students, faculty and staff who are committed to creating a climate of mutual respect that is supportive of one another’s success. Through its curricula and clinical experiences, we purposefully support the University’s goal of diversity, and in particular, work toward an ultimate outcome of best serving the needs of students. Faculty and candidates are expected to demonstrate an understanding of diversity as it relates to planning, instruction, management, and assessment. A WORD ON ETHICS The strength of the university depends on academic and personal integrity. In this course, you must be honest and truthful. Ethical violations include cheating on exams, plagiarism, reuse of assignments, improper use of the Internet and electronic devices, unauthorized collaboration, alteration of graded assignments, forgery and falsification, lying, facilitating academic dishonesty, and unfair competition. Report any violations you witness to the instructor. 020.296 Foreign Gene Expression Lab Schedule 1/9 Names, kits, benches, teams Make LB, autoclave, Pipetting Exercise Overnights of pMC-1871 and pMAL-p 1/10 Minipreps of pMC-1871 and pMAL-p (final form, dry pellet) Problem set 1 due 1/11 Redissolve pellets; Pst-I digest of minipreps 0.8% Agarose Gel EP Problem set 2 due 1/12 Double digest of pMC-1871 and pMAL-p Arctic Phosphatase Digest of pMAL-p 0.6% Agarose Gel; Pour LB+amp plates 1/13 Add Phenol:chloroform to pMal-p digest Add carrier RNA and pMC-1871 digest Complete extraction and ethanol precipitate, dry. 1/16 MLK Birthday – no class 1/17 Redissolve pellet, Blunt ligation; Transform DH5a with ligation product Plate on 5-10 LB+amp + IPTG + X-gal plates 1/18 Select transformants and grow 2 overnights each. Make 0.4 liter LB Medium 1/19 Miniprep of each transformant; Pst I digest & agarose gel Select 9.7 kb plasmid. 1/20 Grow large scale culture, induce with IPTG Harvest by centrifugation, freeze 1/23 Fusion Protein Purification via maltose column. 1/24 Assay Beta-Galactosidase Activity of purification fractions 1/25 SDS-PAGE of Purification; 1/26 Protein Assay 1/27 Final Summary Session; Lab Cleanup; submit lab report Problem set 3 due Problem set 4 due Problem set 5 due Problem set 6 due Problem set 7 due Lab Report Due