Simon Fraser University
Engaging the World

Course Outlines

Spring 2017 - BPK 420 D100

Selected Topics in Kinesiology I (3)

Cellular Physiology Laboratory Course

Class Number: 9089

Delivery Method: In Person

Overview

  • Course Times + Location:

    Jan 4 – Apr 7, 2017: Mon, 1:30–2:20 p.m.
    Burnaby

  • Exam Times + Location:

    Apr 21, 2017
    Fri, 8:30–11:30 a.m.
    Burnaby

  • Prerequisites:

    To be announced in the Undergraduate Schedule of Classes and Examinations.

Description

CALENDAR DESCRIPTION:

Selected topics in areas not currently offered as formal courses within the undergraduate course offerings in the School of Kinesiology. The topics in this course will vary from term to term, depending on faculty availability and student interest.

COURSE DETAILS:

PREREQUISITES: STAT 201 and BPK 305 for BPK Majors or BISC 305 for BISC Majors. Enrolment non-BPK and non-BISC majors requires permission of the instructor.

OUTLINE of SESSIONS:

Sessions Title Content Skills
Week 1 General discussions, lab skills, lab safety, equipment handling and care, pipette skills, aseptic technique, dilutions. Make antibiotic-containing agar plates Pipetting, dilution calculations, aseptic technique
Week 2 Cell counting and introduction to microscopy Tasks: Students receive a sample suspension of cells, and will determine the class average and variance for each sample. Students use Trypan Blue to distinguish live from dead cells. Calculate mean and standard deviation. Use statistical test to decide if three masked samples are different from one another. Students will calculate the number of experiments that can be plated given the sample concentration. Discuss Concepts of dilution, mechanisms of action of trypan blue with respect to cell physiology, discuss concepts of Biosafety when working with cultured cells?  • Pipetting cell suspensions • Microscope work • Data analysis • Experimental planning
Week 3 Molecular techniques I: Bacterial transformation and DNA purification. Tasks: Students will receive two plasmids which will be transformed into E. coli bacteria by heatshock, and will analyse which contains their intended gene of interest. Students will perform DNA isolation from liquid cultures of E. coli bacteria and will quantify DNA yields from their isolates. Discuss how ampicillin works, how transformation works, what is a competent cell. Discuss history of GFP discovery and use of fluorescent marker proteins (protein localization, optical switches, protein-protein interactions with FRET). Discuss elements of plasmid vectors (origin of replication, promoter, resistance gene, MCS, size, copy number and  bicistronic sites and GFP-tags) and reasons for each step in the purification procedure. DNA amplification, purification and isolation. Measuring/weighing chemicals. Aseptic technique.
Week 4 Molecular techniques II: restriction digest, gel electrophoresis, gel imaging and analysis. Tasks: Count colonies from previous week, calculate efficiency. Restriction digestion analysis of DNA isolated in previous week. Discuss: log phase bacterial replication and OD determination, how this amplifies cDNA constructs, restriction enzymes (origin, range, cut efficiency, compatibility, temperature sensitivity), principles of DNA electrophoresis, DNA ladders, DNA visualization (EtBr), super-coiled vs linearized appearance, band densitometry calculation. Vector and gene analysis and restriction digest analysis. Pipetting small volumes accurately, centrifugation. Following protocols. DNA gel analysis.
Week 5 Transfection and Morphological Analysis of cultured smooth muscle cells. Tasks: Characterization of smooth muscle cells (A7R5) using light microscope. Note morphology, density, visible organelles, ultrastructure. Compare differentiated cells versus dedifferentiated cells. Students will transfect cultured cells with isolated plasmids to increase expression of differentiation-inducing transcription factor. Discuss principles of transfection, transfection efficiency, smooth muscle cell morphology, ultrastructure, and phenotype and relevance to vascular injury and angiogenesis. Day after class, visualize and document transfection efficiency via GFP. Lipid-based mammalian transfection, sterile technique, phase contrast microscopy, digital image analysis. Statistical analyses
Week 6 Immunocytochemistry and fluorescence imaging Tasks: Students fix, immunolabel and image transfected cells to assess changing in phenotype / differentiation. Students isolate mRNA from sister cultures in preparation of week 7. Discuss clinical uses of biomarkers giving high profile examples. Discuss limitations of interpretation of fluorescence imaging data. Discuss alternative means to assess protein / mRNA expression levels. Discuss importance of assessing subcellular protein localization and translocation. Principles of randomization and blocked experimental design. Immuno-fluorescence labelling and microscopy, mRNA isolation and methods of mRNA handling. Blocked experimental design
Week 8 Digital droplet PCR: quantification of mRNA levels within cells. Tasks: Use ddPCR to detect changes in expression of myofilament proteins and Klf4 as markers of altered smooth muscle phenotype in response to increased myocardin expression.   Discuss: Discuss principles of PCR (end-point, qPCR and ddPCR). Methods of normalizing mRNA expression to house-keeping genes and selection of house-keeping genes. Discuss limits of interpretation of changes in mRNA relative to changes in protein expression. Discuss methods to assess changes in protein expression. Principles of randomization and blocked experimental design. Precision pipetting. Dilution calculations. Thermal cycling. Digital PCR. Statistical Analysis.
Week 9 Neurophysiology I: earthworm action potential Tasks:  Determine shape, duration, threshold, refractory periods and conduction velocity of earthworm nerve action potential. Students will anesthetize earthworms and place in recording chamber. Student will execute a serial of electrophysiological recordings and analyze acquired traces from iWorx work stations. Discuss: Ethics of animal use in scientific experiments. Principles of neuronal action potential conduction. Principles of electrophysiological recording. Principles of analog-to-digital data conversion and sampling.    Solutions preparation. Animal / invertebrate handling. Electrophysiological recording. Statistical Analysis.
Week10 Smooth muscle regulation I: pharmacological and ionic regulation of rat aorta/earthworm gut contraction Tasks: Students will dissection earthworm gut and perform myography bioassay of a series of pharmacological modulators of spontaneous contractile activity. Discuss: Smooth muscle signal transduction. Uses and limitations of invertebrate models to understand mammalian physiology. Application and interpretations of dose-responses curves. Smooth muscle versus striated muscle length-tension relationship. Student directed, inquiry-based learning
Week11 Final Project Preparation Tasks: Student perform preparation and preliminary tests for final project experiment Discuss: Small group feedback of experimental design. Student directed, inquiry-based learning
Week12 Final Project Execution Tasks: Student perform final project experiment and data collection. Student directed, inquiry-based learning
Week13 Project presentations Tasks: Students present their research findings. Oral presentation skills. Preparation of visual aids appropriate for scientific presentation.

COURSE-LEVEL EDUCATIONAL GOALS:

COURSE OVERVIEW: This course is an advanced theoretical and practical laboratory course in cellular physiological techniques. The course will provide students with sound theoretical understanding and practical hands-on training in fundamental and current biomedical physiology laboratory techniques of DNA manipulation and quantification, fluorescence microscopy, bacteria and mammalian cell culture, bioassays of invertebrate smooth muscle and nerve tissue. The information learned in BPK 305 / BISC 305 is the starting point for discussions and students are encouraged to review their previous course notes and text prior to the start of the course.

Grading

  • Lab sessions (X10) & Graded lab reports(X5) 45%
  • Lab work 10%
  • In-class quizzes 10%
  • Final Quiz 15%
  • Final Project 20%

NOTES:

Final Project: Students will design and undertake a final project over the final lab sessions. This project will require critical analysis of one of the previous lab experiments and a scientific optimization of the procedure with an associated report on the rationale for and efficacy of the optimization methods chosen.

For complete information on BPK's grading policy, please visit:  https://www.sfu.ca/bpk/undergrad_program/faqs/grading_policy.html

REQUIREMENTS:

MISSED CLASS POLICY: Students who miss practical classes due to exceptional circumstances (such as serious illness or compassionate reasons) are required to obtain a physician's certificate, whereby the physician states that you were unable to write your midterm or final on the set date due to a medical condition beyond your control, or other supporting documents in order to obtain consideration in the course.  Such documents must be filed with the Department Chair (via the Biomedical Physiology and Kinesiology office) or Registrar within four calendar days of the date on which the examination was to have been written.   Exceptional circumstances must be approved by the Undergraduate Program Committee in order for a student to receive consideration.  Students must check the class schedule when making course selections.

ACADEMIC HONESTY AND STUDENT CONDUCT: Academic honesty is a condition of continued membership in the University community. Academic dishonesty, including plagiarism or any other form of cheating is subject to serious academic penalty. The University codes of student conduct and academic honesty are contained in policies T10.01 and T10.02 which are available in the Course Timetable and on the Web via http://www.reg.sfu.ca.

Materials

MATERIALS + SUPPLIES:

Provided during lab

REQUIRED READING:

There will be no required course text. Laboratory manual modules curated on CANVAS will contain suggested readings beyond material covered in class.

Department Undergraduate Notes:

The Instructor or the Department reserves the right to curve the grades at the end of the term
For complete information on BPK's grading policy, please visit:  https://www.sfu.ca/bpk/undergrad_program/faqs/grading_policy.html

It is the responsibility of the student to keep their BPK course outlines if they plan on furthering their education.

Registrar Notes:

SFU’s Academic Integrity web site http://students.sfu.ca/academicintegrity.html is filled with information on what is meant by academic dishonesty, where you can find resources to help with your studies and the consequences of cheating.  Check out the site for more information and videos that help explain the issues in plain English.

Each student is responsible for his or her conduct as it affects the University community.  Academic dishonesty, in whatever form, is ultimately destructive of the values of the University. Furthermore, it is unfair and discouraging to the majority of students who pursue their studies honestly. Scholarly integrity is required of all members of the University. http://www.sfu.ca/policies/gazette/student/s10-01.html

ACADEMIC INTEGRITY: YOUR WORK, YOUR SUCCESS