Transcript Goodner2008ASMBioQUEST

What a Great Time to Teach & Do
Research with Undergrads!!
So much data!
So many questions!
So many great tools!
Too few researchers!
*** So much for me
& my students to
do!!!***
As of 1/3/08:
Domain
Published
Ongoing
Archaea
50
68
Bacteria
577
1628
Eucarya
77
891
(+ 114 metagenomes + GEBA plan)
Teaching
Research
Collaborations
Agrobacterium
bv. 2 & 3 strains
(NSF grant w/ 7 partners)
2 Xenorhabdus species
(USDA grant w/ 6 partners)
Ammonifex degensii
(JGI pilot program w/ 10 partners)
Hiram
Genomics
Initiative
Chromohalobacter salexigens
(w/ Purdue Univ. & DOE-JGI)
Sphingomonas elodea
(w/ Monsanto Co.)
Azotobacter vinelandii
(NSF grant w/ 4 partners)
Hiram
Students
High
school
Students
Recruiting
Hiram Genomics Initiative
Agrobacterium
Genome Project
Other Genome Projects
Sphingomonas
elodea
Functional Genomes of
Native Genomics of K84 (bv. 2)
Tumor Strain C58 & S4 (bv. 3)
Genetic/
Survey (biovar 1)
Physical Map
(high
schools)
(Genetics)
Chromohalobacter
Xenorhabdus
Azotobacter
salexigens bovienii & nematophila vinelandii
Genetic/
Physical Map
Genetic/
Physical Map
Gap
Closure
Genetic/
Physical Map
(Genetics)
(Genetics &
high schools)
(Independent
Research)
(Genetics &
Independent
Research)
Gap
Closure
(Independent
Research)
Sequence
Annotation
(MolCell, Genetics,
& Biochem)
Gene
Disruptions
(MolCell &
Independent
Research)
Mutant
Gap
Screens Closure
Sequence
Annotation
(MolCell & (Independent (Genetics &
Independent Research) Independent
Research)
Research)
Sequence
Annotation
(Independent
Research)
Bridging the Teaching-Research Gap
Within Undergraduate Courses
• What prevents us
from incorporating
original research
into the lab
component of
undergraduate
courses?
• Must excite students – move into
independent research projects
• Must excite us
• Must teach key skills & concepts
• Must be doable within time,
space, & budget constraints
• Must be successful as measured
by the norms of science – effective
training for the future,
presentations at conferences, &
publications
Basics of a Genome Project
Subgenomic
Mega-fragments
Genome
Subgenomic
Libraries
6-8X
Sequencing
Coverage
Overlaps in
Small Pieces
to Form Contigs
Gap Closure
Random
Pieces
Shotgun
Genomic
Libraries
Join Large
Pieces into
Sequenced
Genome
Annotation
Functional Genomics
1X
Sequencing
Coverage
Genetic/
Physical
Map
Annotation of
Contig Ends
Opportunity
Sequence Annotation
Subgenomic
Mega-fragments
Genome
Subgenomic
Libraries
6-8X
Sequencing
Coverage
Overlaps in
Small Pieces
to Form Contigs
Gap Closure
Random
Pieces
Shotgun
Genomic
Libraries
Join Large
Pieces into
Sequenced
Genome
Annotation
Functional Genomics
1X
Sequencing
Coverage
Genetic/
Physical
Map
Annotation of
Contig Ends
Annotation Pipeline
0 kb
10 kb
20 kb
•
•
•
•
•
•
Gene finding & operon prediction
Blast & global sequence alignments
Protein domain prediction
Protein localization prediction
Functional prediction
Functional call, linkage to experimental data, &
testable hypotheses (community involvement)
Beyond 1st Pass Annotation
Students as 2nd Pass Annotators
12
C. salexigens
Chromohalobacter
salexigens annotation by
Biochem students to test the
hypothesis that proteins in
halophiles are more acidic
than their homologs in
nonhalophic relatives
E. coli K12
Series4
10
9
8
pI
- PSORT (cellular localization)
- BLAST (homologs in E. coli &
P. aeruginosa)
- MW/pI (pI determination)
P. aeruginosa PA01
11
7
6
5
4
3
3171
3071
3022
2860
2691
2485
2448
2171
2098
1580
1452
1443
1442
1394
1355
1139
1132
941
799
768
766
723
213
2
Figure 2. Isoelectric Points of Outer Membrane Proteins
Opportunity
Testing Bioinformatics Predictions
Subgenomic
Mega-fragments
Genome
Subgenomic
Libraries
6-8X
Sequencing
Coverage
Overlaps in
Small Pieces
to Form Contigs
Gap Closure
Random
Pieces
Shotgun
Genomic
Libraries
Join Large
Pieces into
Sequenced
Genome
Annotation
Functional Genomics
1X
Sequencing
Coverage
Genetic/
Physical
Map
Annotation of
Contig Ends
Functional Genomics
Constructing Gene Disruption Mutants
• Pick genes of interest to you and/or genes with putative
functions that are testable within your course
• Design PCR primers (or have students do so) to amplify an
internal portion of a gene
gene of interest in A. tumefaciens genome
plasmid pCR2.1
cannot replicate
in Agrobacterium
portion
of gene
Cbr
plasmid
portion
of gene
Cbr
• Clone PCR product & confirm by restriction mapping
• Introduce cloned PCR product into wildtype and select for
single crossover gene disruption
Functional Genomics
Constructing Gene Disruption Mutants
• 76 genes disrupted since spring of 2002 by MolCell students
• 49 genes encoding specific enzymes:
multiple genes involved in sucrose metabolism
2 aconitases
4 malate dehydrogenases – only 2 with definable impact
9 penicillin-binding proteins
• 27 genes encoding two component systems (mostly response
regulators):
massive screen of 23 mutants across 54 treatments
(covering 12 different environmental variables)
Functional Genomics
Example = 2 Aconitases in Agrobacterium C58
• One group wanted to look at motility!?
• Motility is one process regulated posttranscriptionally by apo-AcnB in E. coli
wt
acnA-
5
Colony Swarming (cm diameter)
4.5
4
Hour 0
Hour 6.5
Hour 24
Hour 30.5
3.5
3
2.5
2
1.5
1
0.5
0
C58
(LB
0.3)
C58
(LB
0.6)
C58
(LB 1)
C58
(LB
1.5)
acnA(LB
0.3)
acnA(LB
0.6)
acnA(LB 1)
acnA(LB
1.5)
C58
(M9
0.3)
C58
(M9
0.6)
C58
(M9 1)
C58
(M9
1.5)
acnA(M9
0.3)
Strain
(M edium, % Agar)
wildtype A. tumefaciens
from LB plate (pH7)
A. tumefaciens acnA- mutant
from LB plate (pH7)
acnA(M9
0.6)
acnA(M9 1)
acnA(M9
1.5)
Functional Genomics
Forward Genetics Screens
Transposon
mutagenesis
Sequence off of Tn end to
identify mutated gene
Mutant screening & characterization
Recovery of Tn insertion site
Forward Genetic Screens
High School Students Can Do It
• Real world = multiple classes since
2002 from 5 area high schools
• Auxotrophs are easy to screen & connect to larger issues of
metabolism & nutrition - learn bacterial genetics,
mutagenesis, connect genes to enzymes to pathways
• If needed, college students physically map insertions restriction mapping of DNA
• obtain sequences at insertion sites - learn DNA sequence
analysis, connect genotype to phenotype
Forward Genetic Screens
Hiram Genomics Academy
• 79 students from high schools in
OH, PA, MI, & IN spread over 5
summer sessions in 2006 & 2007
• Each session lasted 3-5 days
• Students generated mutants, screened for phenotypes,
recovered Tn insertion sites for sequencing, & learned some
bioinformatics
• high school students + 11 Hiram students generated &
screened 10,500 mutants for 10 different phenotypes, &
identified 110 mutants worthy of further study