Expansion of Gonzaga University’s phage research program
Marianne Poxleitner, Kirk Anders, Amanda Braley, and Ann-Scott Ettinger
Department of Biology, Gonzaga University, Spokane, WA 99258
The Challenge
Phage Discovery Lab
Genetics Lab
Fall Family Phage Symposium
Provide all introductory biology students at Gonzaga
with a lab course that provides a research experience
The In Situ portion of the phage course was taught to ~360
incoming science freshmen as the introductory biology lab
course serving many majors:
The In Silico portion was taught as two modules within a
Genetics Laboratory course (BIOL 207L). This year, ~90
students participated.
Select students presented their research during a mini
phage symposium scheduled during the annual “Fall
Family Weekend” activities.
After observing the student enthusiasm and outcomes of the first two
years of the SEA PHAGES course, our colleagues asked if we could
modify the course to serve as the lab for our large introductory
biology course in our new curriculum. But how to do it in one day per
week? What about genome annotation? Can we keep the “magic” at
an industrial scale?
Our Strategy
•
•
•
•
•
•
•
•
•
Biology
Biochemistry
Human Physiology
Nursing
Premed (non-majors)
Computer Science (a few)
Implementation
We converted the In Situ and In Silico portions of a SEA PHAGES
course into a progression of experiences that are vertically integrated
in our curriculum.
Figure 3. Lots of enrichment cultures
shaking at room temperature.
Table 1. Semester lab activity schedule by week.
Week
1
The Phage Discovery Lab serves a large number of freshmen
students, both science and health science majors.
2
Plate enrichment supernatant in dilution series
3
7
Achieve these activities:
Test putative plaques
Plaque-purify at least three rounds
Determine titers of plaque lysates
Flood “web” plate to produce medium titer lysate
Titer medium titer lysate
(a few exceptional students prepare high titer lysate)
8
Mount samples for transmission electron microscopy
9
DNA prep
4
The Genome Annotation and Phage DNA Cloning modules in the
200-level Genetics Laboratory course serve all biology and
biochemistry majors.
5
6
The Advanced Phage Research lab serves a small number of
interested 3rd and 4th year students.
In addition to extra supplies from the SEA, we obtained funding from
the NSF-TUES program and from Gonzaga University to supplement
lab fees and equip laboratory classrooms to teach these courses.
Phage
Research Lab
Activity
Collect soil, inoculate enrichment culture
90 students/year
Phage Discovery Lab
360 introductory students/year
Figure 1. Gonzaga University’s three tiered phage research program.
• One freshman from the Phage Discovery Lab presented
background materials and details about isolation,
purification, and analysis of her phage
Figure 6. Genetics students showing attitude.
Genome Annotation Module (5 weeks)
• Finding genes in DNA: 6-frame translations, ORFs, RBS, coding
potential
• Learn button-pushing in DNA Master. Annotate 1-2 genes as a class
• Annotation in teams of two pairs. Each team annotates 6-10 genes
• Class presentations of gene calls and functions
• Teams work on exploratory projects (find other elements/repeats,
compare sequences and phylogenies, propose own question, etc.)
A
• A previous SEA PHAGES student presented bioinformatics
results from the previous year on the sequenced phage
Phoxy
• Six freshmen presented Phage Discovery posters
assembled the previous semester during the Fall Family
Weekend poster session (usually reserved for
upperclassmen presenting faculty research)
• 65 attendees were able to see the cohesive nature of our
phage program. Students got to see what phage fun was
was to come and parents observed students presenting
work done as part of our Biology curriculum
B
10
DNA prep
11
Restriction digest
12
Agarose gel electrophoresis
13
Restriction fragment gel analysis
• MTL DNA prep again, Sal I digest, ligate to pBluescript
14
Student poster presentations
• Transform competent E. coli, plate to Amp + X-gal plates
15
Final exam and assessment
• Isolate plasmids from transformants, measure insert size
Phage DNA Cloning and Sequencing Module (5 weeks)
• Students recover their own phage from archived lysate
• Send clone for Sanger sequencing with T3 and T7 primers
•
•
•
•
•
•
•
12 lab sections fall semester, and 12 spring semester
Meet one day a week for 3 hours
16 students per lab work in pairs to isolate phage
Taught by faculty
Hire teaching assistants from previous semesters
Utilized lab technique videos and online quizzes to save class time
Incubate cultures at room temp to slow growth and facilitate large
numbers of plates
• Purification shortcuts include streak plates outside of class time
• Use MTL for TEM and DNA isolation
Assessment
• SEA CURE -- compare to full-year courses
• Pre/post concept test
• Open-ended survey to probe scientific thinking, curiosity, and
enthusiasm (we are studying these for patterns, hints, and ideas
about outcomes we might try to measure in the future)
• Analyze sequence, assign tentative cluster, propose which phage
genomes should be sequenced in future
Advanced Phage Research Lab
To begin Fall 2014. Students who have completed Genetics Lab
may apply for this course, and take it multiple times if they wish.
Purpose
•
want to stay involved in phage research -or-
•
want research experience in hopes of eventually getting an
undergraduate research position
• Run like a research group. Research outside of class time is
required.
• A mixture of faculty-organized and student-generated projects to
choose from
• Molecular and/or bioinformatics methods to be used
• Work in small teams or individually
• Students write proposals, design experimental approaches, present
during informal lab meetings, formal presentations at end
Figure 2. Students participating in Phage Discovery Lab.
Figure 5. Fall 2012 shipment. That’s a lot
of boxes!
Figure 9. Finding plaques during Faculty Phage training.
Faculty Phage Training
Enhance faculty understanding of the Phage Discovery Lab
goals
• Offered summer training to faculty at local universities and community
colleges to share materials and strengthen the research oriented goals of
the course, and discourage teaching it like a “cookie- cutter” lab.
• Assembled a “Faculty Phage” training manual to highlight the research
goals of each period and suggest ways to facilitate discussions and avoid
lectures.
• Additional training with faculty teaching freshman Phage Discovery labs at
Gonzaga will focus on reducing lecturing and encourage using a “lab
meeting” type approach in the classroom.
• Reinforce these goals with weekly meetings to make sure everyone is on
task and teaching in a manner that is in keeping with the HHMI vision of the
SEA PHAGES course.
To provide opportunity for students to participate in independent-style
research. These are students who:
Tentative plan
Figure 4. Students doing a restriction
enzyme digest activity
• A senior presented cluster typing and sequencing results for
the Genetics Lab
Figure 7. Blue/white screening (A) and Sanger sequences (B) from molecular biology module.
10-20 students/yr
Phage Genomics and
Molecular Biology Modules
Biology majors
Biochemistry
Premed (non-majors)
Acknowledgments
Lu Barker, Kevin Bradley (SEA HHMI) and Debbie Jacobs-Sera, Welkin Pope,
Dan Russell, Graham Hatfull (Pittsburgh Bacteriophage Institute) for training,
assistance, and encouragement
David Asai, Cheryl Bailey, Lu Barker, and HHMI for supplies and other
encouragement and support
NSF TUES program for equipment and support
Gonzaga University for support and enthusiasm
Leslie Jaworski and David Lopatto for CURE survey results