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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