joanna klein, ph.d. northwestern scholarship symposium may 4, 2012
TRANSCRIPT
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Joanna Klein, Ph.D.Northwestern Scholarship Symposium
May 4, 2012
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What are Bacteria?Single celled microorganism Friend or Foe?
Friend: health, environment, industryFoe: cause a variety of infectious diseases
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Cellulophaga lyticaMarine bacteriumIsolated from beach mud
near Limon, Costa Rica in 1969
http://www.infoplease.com/atlas/centralamerica.html
http://travel.yahoo.com/p-travelguide-482616-limon_vacations-i
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Cellulophaga lyticaGram negative FilamentousYellow pigmentationExhibits gliding motility
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Gliding Motility
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Cellulophaga lyticaMember of the Cytophaga-Flavobacterium-
Bacteroides (CFB) group of bacteriaPoorly characterized branch
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Phylogenetic tree of Bacteria
ProteobacteriaE. coli,
Salmonella, Bordetella, Helicobacter, Vibrio
FirmicutesStaphylococcus,
Streptococcus, Lactobacillus, Clostridium
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Cellulophaga lyticaTarget organism in the Genomic Encyclopedia of
Bacteria and Archaea (GEBA) Research Program of the Department of Energy/Joint Genome Institute
GEBA organisms 100 representative
organisms from each of the branches
Organisms with potential energy applications
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Biofuel productionC. lytica produces a variety of
enzymes that may have applications in biotechnology and biofuel production
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Deconstruction by C. lyticaC. lytica contains many polysaccharide degrading
enzymes Polysaccharides
Large molecules that store energy or provide structure Carbohydrates/starches Cellulose in plant cell walls
Enzymes break down polysaccharides into simple sugars that that can be fermented to produce energy
Polysaccharide degrading enzymes3 cellulases3 fucoidases1 xylosidase
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Plant Cell Wall
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Ethanol productionEthanol produced as a byproduct of starch
degradation and subsequent fermentationWell developed technologyEnzymes digest starch into simple sugars
which are readily fermented by known microorganisms to produce ethanol
Issues…
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Cellulosic ethanol productionGoal is to use the cellulose biomass found in
plant cell walls of leaves and wood to produce ethanolProblems to overcome:
Lignin, also found in cell wall, hinders digestion of cellulose from wood
Enzymes that digest cellulose into simple sugars are poorly understood
Organisms that ferment these simple sugars to produce ethanol are poorly understood
Can C. lytic help achieve this goal?
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Why study C. lytica?Model organism to understand the CFB
group betterContribute to biofuel research and
applications
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Genome Annotation of Cellulophaga lyticaOne way to understand more about the life
processes of C. lytica is through a study of its genome.
GenomeAll of the genetic material, DNA, of an
organismDNA is made up 4 smaller molecules known as
the bases A,C,G &T
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Sequencing genomesWe can easily determine the entire DNA
sequence of an organism – it’s genome.DNA sequencing technology has developed
rapidly since the human genome project, completed in 2003Took 13 years to complete, involved 100’s of
researchers around the globe, and cost a total of of $2.7 billion
Entire 3 billion base-pair sequence is available in a public database
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Genome projectsCurrently, there are more than 3000
complete or nearly complete genome sequences of microbes available.
Over 1200 genome sequencing projects in higher organisms (plants, animals, fungi, protists)
The complete genome of Cellulophaga lytica was sequenced by the DOE and published in 20113,765,936 bases
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Genome Projects
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Computer annotation of C. lyticaNumber of genes and predicted function of
each gene product.
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TATCAAAGAGATGATTGAGAACTGGTACGGAGGGAGTCGAGCCGGGCTCACTTAAGGGCTACGACTTAAC GGGCCGCGTCACTCAATGGCGCGGACACGCCTCTTTGCCCGGGCAGAGGCATGTACAGCGCATGCCCACA ACGGCGGAGGCCGCCGGGTTCCCTGACGTGCCAGTCAGGCCTTCTCCTTTTCCGCAGACCGTGTGTTTCT TTACCGCTCTCCCCCGAGACCTTTTAAGGGTTGTTTGGAGTGTAAGTGGAGGAATATACGTAGTGTTGTC TTAATGGTACCGTTAACTAAGTAAGGAAGCCACTTAATTTAAAATTATGTATGCAGAACATGCGAAGTTA AAAGATGTATAAAAGCTTAAGATGGGGAGAAAAACCTTTTTTCAGAGGGTACTGTGTTACTGTTTTCTTG CTTTTCATTCATTCCAGAAATCATCTGTTCACATCCAAAGGCACAATTCATTTTGAGTTTCTTTCAAAAC AAATCGTTTGTAGTTTTAGGACAGGCTGATGCACTTTGGGCTTGACTTCTGATTACCCTATTGTTAAATT AGTGACCCCTCTTAGTGTTTTCCTGTCCTTTATTTCGGAGGACGCACTTCGAAGATACCAGATTTTATGG GTCATCCTTGGATTTTGAAGCTTATAACTGTGACAAAAAATGTGAAGGGAAGAGATTTGAAACATGTGGA AGGAAAAGTGAGTGCAGACTATAAACTTCCAAAAAGACAAGCCCAAAATACACCTAAACGTTATGTCAGA TTATTTTGTTAAAATCAGTTGTTAGTGACGTCCGTACGTTAATAGAAAAAAGAATGCTTCAGTTTGGAGT GGTAGGTTTCTAGAGGGATTTATTGTGAAAGTATAAACTATTCAGGGCAATGGGACTGAGAGAACAGTGG GTAGAAAGGACCACTGAAGGAAAGGAAGAGAATTGGAAGGTAGATGAAAGAAGGAGCAAGAACCTGGGGTGTTTTTTCCTTTTCACTTGTAATAGTAGTAACAGAAGCAATGGCAGACTGGCTTTTGTTTCTACTGTGT TAGAATGAATTGACAGGACAACTGGGCCTATTATTGTACTGTGCCAGAATACTGTAAAACAAAACTAAAC ATACTAGCTTGGTGGCTTGTAATTAATTACTTAAGTGGAGATTTTTATTTTTTTTTTATTTTTTTTTTAG ACGGAGTCTCACTTTGTCACCCAGGCTGGAGTGCAGTGGCGCGATCTCAGCTGACTGCAACCTCCTCCTC
Cellulase
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Process of annotationAutomatic annotation - done automatically
using computer software35% of computer generated annotations are wrong or
are missing information due to limitations of computer algorithms
Manual Annotation – humans analyze the information generated by computers and make corrections as necessary. Labor intensive and time consumingSolution: Train students to participate in the
process
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• IMG-ACT is a toolkit of online gene and genome analysis programs.
• Using IMG-ACT, students annotate genomes• provide human expertise necessary for
accurate, up-to-date, reliable annotation• Students contribute to the scientific
community and learn biological concepts through participating in original research
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JGI Genome Annotation Workshop Walnut Creek, CA January 2011
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Genome annotation of C. lytica at NWC39 NWC students have participated in this
research endeavorScience Research Institute, Summer 2011Genetics, Fall 2011Microbiology, Spring 2012
15 genes have been fully annotated10 genes have been partially annotated
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Restriction endonuclease type IWhat is the amino acid sequence of the
protein encoded by this gene?Used Integrated Microbial Genomes (IMG)
databaseAmy Knight and Allison Lothe
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DNA topoisomerase IIIHow does this
protein compare to the sequence of other proteins?
Used BLAST program
Libby Nelson and Chelsey Fiecke
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RNA polymerase sigma subunit 24What are key functional amino acid residues in the
protein?Web Logo ProgramSilas Baalke and Laura Torgerson
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DNA Replication Protein AWhat enzymatic pathway is the protein involved in?Used KEEG Pathway databaseMarie Abeler and Gabe Jefferson
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-galactosidaseWhat pathway is this enzyme found in?KEEG databaseDaniel Plack, Michael Lowry
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Prolyl-tRNA synthaseWhat is the 3D
structure of similar proteins?
ProteinDataBank (PDB)
Sarah Ivanca and Victoria Hanson
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NusA, B, G anti-termination factors
Where is the gene in relation to other genes?Used Gene neighborhood feature of IMGMatt Takata and Zach Fredman
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RNase HWhat reaction does the enzyme catalyze?Used Metacyc databaseChelsey Fiecke
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Elongation Factor TsHow closely related is this protein to proteins
in other bacteria?Used Phylogeny FR programEllen Chae, Holly Tomaz
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Cytochrome C oxidase subunit 3Where is this protein located in the cell?TMHMM algorithmAlannah Pratt, Michael Lowry, SRI high school
students
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MutSAre there paralogs of this gene?IMG database queryRyan Bradbury and Luke Delain
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RNA polymerase sigma-70 factorWas this gene named properly?Multiple lines of evidence used to change
name to RNA Polymerase anti-sigma 70 factorCamaren Terrill and Ben Sorenson
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Future work3,348 genes left to annotate!
Special interest in: Polysaccharide degrading enzymes Motility proteins Proteins with unknown function
Study the function of interesting genes in the lab
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Genome Projects
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AcknowledgementsNWC students who have participated in this
research.Genetics, Microbiology and SRI coursesResearch students Steven Erickson and Andy
Jaeger Northwestern College for providing the
opportunity and support for the sabbatical during which this project was initiated.
Additional funding received from a 2010-2011 Faculty Development Grant