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Functional Genomics through Complementation in the Classroom Steven Slater
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 ThemW
e 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 numberAmp resistant to avoid outgrowth after transformationsacB gene provides counter-selectable marker to remove backgroundArabinose-inducible expression of gene-of-interestCloning site flanked by NotI sitesDesigned for ligase-independent cloning
sacB5’GAATTCGACAAGAGCGGCCGC ATGAACATCAAAAAGTTTGC3’CTTAAGCTGTTCTCGCCGGCG TACTTGTAGTTTTTCAAACG
no As
no As
vecto
r
. . . .
.
. . . .
. no Ts
no Ts
. . . .
. . . . .
.
NotI
weak RBS
vectorNotIERI
GGACAATTAACAGTTAACAAATAA GCGGCCGCTTGGTGTTTCTAGAATCATG -3’CCTGTTAATTGTCAATTGTTTATT CGCCGGCGAACCACAAAGATCTTAGTAC-5’
proC5’ CGACAAGAGCGGCCGC ATGGAAAAGAAAATCGGTTTTATTGGC3’ GCTGTTCTCGCCGGCG TACCTTTTCTTTTAGCCAAAATAACCG
CTCAGCAAATCCTGATGA GGCCGCTTGGTGTT 3’ GAGTCGTTTACCACTACT CCGGCGAACCACAA 5’
Ligation Independent Cloning
5’GAATT3’CTTAAGCTGTTCTCGCCGG
vecto
r
NotI
vector
ERI
XbaI
proC5’ CGACAAGAGCGGCCGC ATGGAAAAGAAAATCGGTTTTATTGGC 3’ ACCTTTTCTTTTAGCCAAAATAACCG
CTCAGCAAATCCTGATGA 3’ GAGTCGTTTACCACTACT CCGGCGGAACCACAA-5’
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.
5’-GAATTCGACAAGAGC -3’3’-CTTAAGCTGTTCTCGCCGG-5’
vecto
r
NotI
vectorNotIERI XbaI
digest vector with NotI
Treat vector and insert with T4 DNA Polymerase
dTTP
insert
dATP
dTTP
dATP
5’-GGCCGCTTGGTGTTTCTAGA-3’3’- CGAACCACAAAGATCT-5’
5’-GGCCGCTTGGTGTTTCTAGA 3’3’- TCT5’
5’-CGACAAGAGCGGCCGCATGGAAAAGAAAATCGGTTTTATTGGC -3’ 5’-AACACCAAGCGGCCGAAAGTCATCAGGATTTGCTGAGT-3’
Proline Genes
proA
proB
proC
Arginine Genes
argA
argB
argC
argD
argE
argF
argG
argH
argI
carA
carB
Our first kits are being built around Amino Acid Biosynthesis Pathways
Aspargine/ Isoleucine
Genes
asnA
asnB
ilvA
ilvC
ilvD
ilvE
ilvBN
ilvGM
ilvIH
Glutamine/ Ammonia Assimilation
Genes
glnA
glnB
glnD
glnE
glnG
glnL
ropN
Alanine Genes
alr
dadB
dadX
avtA
M9 (No Ara) M9+Arginine (No Ara) M9+Arabinose
argE (atu3398)
argE (atu5479)
wt
argE
argE +argE K12 Pos. Control
Expt.argE +(atu3398)
wt
wt
wt wt wt
argEargE
Neg. Control argE +sacB
argE argE argE
Expt.argE +(atu5479)
argE +argE K12 Pos. Control
Neg. Control argE +sacB
argE +argE K12 Pos. Control
Neg. Control argE +sacB
Expt.argE +(atu3398)
Neg. Control argE +sacB
argE +argE K12 Pos. Control
Expt.argE +(atu3398)
Expt.argE +(atu5479)
argE +argE K12 Pos. Control
Neg. Control argE +sacB
Expt.argE +(atu5479)
argE +argE K12 Pos. Control
Neg. Control argE +sacB
A. tumefaciens C58 argE Complementation Assay
(Experiment done by SPU undergraduate Jake Sharp)
The Arginine Biosynthetic Pathway
From: Xu, et. al. 2007. Microbiol. Mol. Biol. Rev. 71: 36-47.
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 our 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
The Benefits of this Approach
Contributors
NSFFunding by:
Dr. Steven Slater – The University of Wisconsin-Madison DOE Great Lakes Bioenergy Research Center
Dr. Derek Wood – Seattle Pacific UniversityDr. Katey Houmiel – Seattle Pacific University
Dr. David Rhoads – University of Arizona
Dr. Brad Goodner – Hiram College
The Mesa High School Biotechnology AcademyXan SimonsonAmanda GrimesKen Costenson
