Functional Genomics through Complementation in the Classroom

1. Functional Genomics through Complementation in the Classroom American Society for Microbiology May 24, 2010

2. Biology education lags far behind biological research. • The Education that science students are experiencing is lagging seriously behind “how biologists design, perform and analyze experiments.” (National Research Council) • “Biology students are generally poorly prepared to perform the hands-on lab work required in research settings.” (Battelle, 2003 study for the Flinn Foundation)

3. “Practice like you play…” • This age-old coaching wisdom also applies to other types of education • Students should learn experimental science by doing experimental science • Our Philosophy: Whenever possible, classroom experimentation should be real research. That is, the experiments should be designed to produce novel results that contribute to the body of scientific knowledge

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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.

10. 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

11. Ligation Independent Cloning no As 5’CGACAAGAGCGGCCGC ATGGAAAAGAAAATCGGTTTTATTGGC 3’ GCTGTTCTCGCCGGCGTACCTTTTCTTTTAGCCAAAATAACCG CTCAGCAAATCCTGATGAGGCCGCTTGGTGTT 3’ GAGTCGTTTACCACTACT CCGGCGAACCACAA5’ proC insert no As . . . . . . . . . . . . . . . . . . . . weak RBS no Ts GGACAATTAACAGTTAACAAATAA GCGGCCGCTTGGTGTTTCTAGAATCATG -3’ CCTGTTAATTGTCAATTGTTTATT CGCCGGCGAACCACAAAGATCTTAGTAC-5’ 5’GAATTCGACAAGAGCGGCCGCATGAACATCAAAAAGTTTGC 3’CTTAAGCTGTTCTCGCCGGCG TACTTGTAGTTTTTCAAACG sacB vector vector ERI NotI NotI no Ts 5’-GGCCGCTTGGTGTTTCTAGA-3’ 3’-CGAACCACAAAGATCT-5’ 5’-GAATTCGACAAGAGC-3’ 3’-CTTAAGCTGTTCTCGCCGG-5’ digest vector with NotI vector vector NotI XbaI ERI NotI dTTP Treat vector and insert with T4 DNA Polymerase XbaI 5’-GGCCGCTTGGTGTTTCTAGA3’ 3’-TCT5’ vector dATP 5’ CGACAAGAGCGGCCGC ATGGAAAAGAAAATCGGTTTTATTGGC 3’ ACCTTTTCTTTTAGCCAAAATAACCG CTCAGCAAATCCTGATGA 3’ GAGTCGTTTACCACTACT CCGGCGGAACCACAA-5’ proC dATP 5’GAATT 3’CTTAAGCTGTTCTCGCCGG vector ERI NotI 5’-CGACAAGAGCGGCCGCATGGAAAAGAAAATCGGTTTTATTGGC-3’ 5’-AACACCAAGCGGCCGAAAGTCATCAGGATTTGCTGAGT-3’ dTTP 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.

12. Our first kits are being built around Amino Acid Biosynthesis Pathways Proline Genes proA proB proC Arginine Genes argA argB argC argD argE argF argG argH argI carA carB Aspargine/ Isoleucine Genes asnA asnB ilvA ilvC ilvD ilvE ilvBN ilvGM ilvIH Glutamine/ Ammonia AssimilationGenes glnA glnB glnD glnE glnG glnL ropN Alanine Genes alr dadB dadX avtA

13. A. tumefaciens C58 argE Complementation Assay (Experiment done by SPU undergraduate Jake Sharp) M9 (No Ara) M9+Arginine (No Ara) M9+Arabinose Neg. Control DargE +sacB Neg. Control DargE +sacB Neg. Control DargE +sacB wt DargE DargE DargE wt wt argE (atu3398) DargE +argE K12 Pos. Control DargE +argE K12 Pos. Control DargE +argE K12 Pos. Control Expt. DargE +(atu3398) Expt. DargE +(atu3398) Expt. DargE +(atu3398) Neg. Control DargE +sacB Neg. Control DargE +sacB Neg. Control DargE +sacB DargE DargE DargE wt wt wt argE (atu5479) DargE +argE K12 Pos. Control DargE +argE K12 Pos. Control DargE +argE K12 Pos. Control Expt. DargE +(atu5479) Expt. DargE +(atu5479) Expt. DargE +(atu5479)

14. The Arginine Biosynthetic Pathway From: Xu, et. al. 2007. Microbiol. Mol. Biol. Rev. 71: 36-47.

15. There’s another option for the argE Reacton

16. 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

17. 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

18. Contributors NSF 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: