Functional Genomics through
Complementation in the Classroom
Steven Slater
Some Issues with Research in
the Classroom
Difficult for instructors to
identify/implement novel research projects
each semester/year
Planning/preparation time must be efficient
(minimal) and easily accomplished by busy
faculty or teaching assistants
Experiments must be well defined and
capable of producing clear outcomes
Should address multiple scientific topics
within a series of experiments
How We are Working to Solve Them
We have designed the core of a curriculum, based on
genetic complementation of defined E. coli mutants,
that enables true experimentation in the classroom
The modular format allows testing completely different
genes every semester but does so with repetitive sets of
protocols and materials
Consistency makes it possible for busy faculty to
perform actual research in the classroom without having
to prepare de novo labs every semester
Why use Complementation as a
basis for curriculum modules?
In many cases, it provides a clear “life or
death” result that can be easily interpreted.
 Although intermediate phenotypes (e.g. slow
growth) can be distinguished.
It lends itself to testing thousands of different
types of genes.
 Assays can be simple (survival or colorimetric),
complex (GC analysis of metabolites) or anything
in between
Why use Complementation as a
basis for curriculum modules?
The framework enables integration of many
techniques and genetic principles
 Bioinformatics (from simple BLAST through programming)
 Auxotrophy vs. prototrophy; Epistasis
 Basic molecular techniques such as PCR, cloning, selection,
restriction endonuclease mapping, etc.
 Gene induction and regulation
 If desired, enzymatic analysis
Each experiment provides functional data that can be
used to update annotation and construct publications
Why use Complementation as a
basis for curriculum modules?
Perhaps most importantly, it lends itself to
highly repeatable experiments that are all
variations on a theme
The vectors, techniques, and (most)
instructional materials are consistent from
semester to semester
The primary changes each year involve the
particular pathway under investigation and
the choice of genes to test
We are Enabling the System
Through Curriculum “Kits”
We aim to combine the engagement of original
research with the straightforward techniques typical
of “kits”, such as those for cloning GFP
Each kit contains:
 A defined E. coli mutant and isogenic WT strain
 A cloning vector
 A positive-control plasmid containing the E. coli version of
the gene
 Complete protocols
 Background information on the experiment
 Support via a web site for downloading information, asking
questions, uploading results, and connecting with other
groups performing similar or identical sets of experiments.
We developed a specific vector for the program
Broad host-range (pBBR origin)
Low copy number
Amp resistant to avoid outgrowth after transformation
sacB gene provides counter-selectable marker to remove background
Arabinose-inducible expression of gene-of-interest
Cloning site flanked by NotI sites
Designed for ligase-independent cloning
Ligation Independent Cloning
5’-GAATTCGACAAGAGC
-3’
3’-CTTAAGCTGTTCTCGCCGG-5’
ERI
NotI
CTCAGCAAATCCTGATGA GGCCGCTTGGTGTT 3’
GAGTCGTTTACCACTACT CCGGCGAACCACAA 5’
sacB
GGACAATTAACAGTTAACAAATAA GCGGCCGCTTGGTGTTTCTAGAATCATG -3’
CCTGTTAATTGTCAATTGTTTATT CGCCGGCGAACCACAAAGATCTTAGTAC-5’
NotI
digest vector with NotI
Treat vector and insert with T4 DNA Polymerase
dATP
. . .. .
weak
no Ts
RBS
5’GAATTCGACAAGAGCGGCCGC ATGAACATCAAAAAGTTTGC
3’CTTAAGCTGTTCTCGCCGGCG TACTTGTAGTTTTTCAAACG
ERI
NotI
proC
. . .. .
no As
. . .. .
5’ CGACAAGAGCGGCCGC ATGGAAAAGAAAATCGGTTTTATTGGC
3’ GCTGTTCTCGCCGGCG TACCTTTTCTTTTAGCCAAAATAACCG
. . . . .
insert
no As
no Ts
5’-GGCCGCTTGGTGTTTCTAGA-3’
3’CGAACCACAAAGATCT-5’
NotI
XbaI
dTTP
XbaI
5’-GGCCGCTTGGTGTTTCTAGA 3’
3’TCT5’
5’ CGACAAGAGCGGCCGC ATGGAAAAGAAAATCGGTTTTATTGGC
CTCAGCAAATCCTGATGA 3’
3’
ACCTTTTCTTTTAGCCAAAATAACCG proC GAGTCGTTTACCACTACT CCGGCGGAACCACAA-5’
5’GAATT
3’CTTAAGCTGTTCTCGCCGG
ERI
NotI
dTTP
dATP
5’-CGACAAGAGCGGCCGCATGGAAAAGAAAATCGGTTTTATTGGC -3’
5’-AACACCAAGCGGCCGAAAGTCATCAGGATTTGCTGAGT-3’
T4 DNA Polymerase 3’
5’ exonuclease digests DNA until the first specified nucleotide (A or T) is reached. T4 DNA Polymerase idles at the A or T
since the enzyme defaults to the polymerizing activity when dATP or dTTP is supplemented into the respective reaction.
Our first kits are being built around Amino Acid
Biosynthesis Pathways
Proline Genes
Arginine Genes
proA
argA
proB
argB
proC
argC
argD
Alanine
Genes
alr
dadB
dadX
avtA
argE
argF
argG
argH
argI
carA
carB
Aspargine/
Isoleucine
Genes
asnA
asnB
ilvA
ilvC
ilvD
ilvE
ilvBN
ilvGM
ilvIH
Glutamine/
Ammonia
Assimilation
Genes
glnA
glnB
glnD
glnE
glnG
glnL
ropN
A. tumefaciens C58 argE Complementation Assay
(Experiment done by SPU undergraduate Jake Sharp)
M9 (No Ara)
DargE
M9+Arginine (No Ara)
Neg. Control
DargE +sacB
wt
DargE
M9+Arabinose
Neg. Control
DargE +sacB
Neg. Control
DargE +sacB
wt
DargE
wt
argE
(atu3398)
DargE +argE K12
Pos. Control
DargE
argE
(atu5479)
Expt.
DargE +(atu3398)
Neg. Control
DargE +sacB
DargE
Expt.
DargE +(atu3398)
Expt.
DargE +(atu5479)
DargE +argE K12
Pos. Control
DargE +argE K12
Pos. Control
Neg. Control
DargE +sacB
DargE
Expt.
DargE +(atu3398)
Neg. Control
DargE +sacB
wt
wt
wt
DargE +argE K12
Pos. Control
DargE +argE K12
Pos. Control
Expt.
DargE +(atu5479)
DargE +argE K12
Pos. Control
Expt.
DargE +(atu5479)
The Arginine Biosynthetic Pathway
From: Xu, et. al. 2007. Microbiol. Mol. Biol. Rev. 71: 36-47.
The Benefits of our Approach
It motivates learning
 Increases Enthusiasm of Students and Instructors
 Provides a sense of accomplishment
 Combines theoretical knowledge with the
practical application of skills
It can lead to individual research projects
coming out of the classes
It provides functional data to the scientific
community to support gene annotation
The Benefits of this Approach
Instructors engage more with the program content
and, hence, with the students
 Research programs can be easily integrated into standard
teaching practices.
 Instructors at the High School, Community College and
Undergraduate University levels are impacted.
Provides flexibility for instructors
 Instructors can enter program at any “degree of difficulty”
 Instructors can work on any organism or pathway, or
integrate with one of our ongoing projects
 Data collection, validation, and manuscript preparation are
enabled by a network of institutions focused on the same
approach, and often the same organism
Contributors
Dr. Steven Slater – The University of Wisconsin-Madison
DOE Great Lakes Bioenergy Research Center
scslater@glbrc.wisc.edu
Dr. Derek Wood – Seattle Pacific University
Dr. Katey Houmiel – Seattle Pacific University
houmk@spu.edu
Dr. David Rhoads – University of Arizona
Dr. Brad Goodner – Hiram College
The Mesa High School Biotechnology Academy
Xan Simonson
Amanda Grimes
Ken Costenson
Funding by:
NSF