A Developmental Approach to Integrating Bioinformatics with Laboratory Experiments in Several Undergraduate Courses

Jeff Newman Lycoming College April 29, 2000

Project Overview

Molecular Lab + Lasergene Bioinformatics + Chime Molecular Visualization • Bio110 - Introductory Biology - Mr. Green Genes • Bio222W - Genetics - Human Clotting Factor IX Cloning • Bio321 - Microbiology - Unknown Microbe Identification • Bio437 Molecular Biology gene and cDNA.

Comparison of mouse clotting factor IX • Other 400-level - Cell Biology, Biochemistry – Integrated Informatics Project

Mr. Green Genes Wet Lab

• Week 1 - pGLO Plasmid DNA Isolation (CTAB-boiling/lysozyme method) • Week 2 - Transformation of plasmid. Chime and Lasergene Demonstrations.

• Week 3 - Restriction Enzyme Digestion, Gel Electrophoresis

Mr. Green Genes Bioinformatics

• Step by step instructions walk students through analysis • Retrieve and Display the pGLO Plasmid DNA Sequence • Identify Genes on the Plasmid – Identify ORFs (concepts of start, stop codons, reading frames, antiparallel strands) – Translate ORFs – Search GenBank • Create a Restriction Map

BLAST Sequence Similarity Search (Basic Local Alignment Search Tool)

BLAST Sequence Similarity Search (Basic Local Alignment Search Tool)

Factor IX Cloning Wet Lab (lab meets 3hr/week)

• Week 1 – Isolate cheek cell DNA, set-up PCR, agarose gel • Week 2 – Clean up PCR product, cut PCR product and plasmid vector with restriction enzyme. Analyze products by agarose gel electrophoresis.

• Week 3 - Ligate DNAs, Transform into E.coli

. Screen via  -complementation.

Demonstrate bioinformatics exercise.

• Week 4 - Isolate Plasmid DNA.

• Week 5 - Restriction enzyme digestion of plasmids and agarose gel electrophoresis.

Factor IX Cloning Bioinformatics

• Learn about Factor IX from student-made

Interactive

Chime tutorial.

• Obtain Factor IX gene and cDNA Sequences from the GenBank Database.

Factor IX Cloning - Bioinformatics

• Examine human Factor IX intron-exon organization.

• Identify fragment to be amplified.

• Align Factor IX DNA Sequences.

Factor IX Cloning - Bioinformatics

• Compare Factor IX Protein Sequences • Construct Recombinant DNA Sequence. • Map the Recombinant Plasmids

Unknown Microbe Identification Wet Lab (lab meets 2 x 2hr/week)

• Week 1 - Obtain sample from environment • Week 2 - Pure culture, gram stain • Week 3 - Carbon metabolism • Week 4 - Nitrogen metabolism • Week 5 - Oxygen requirement, exoenzymes, Bergey’s Manual • Week 6 - PCR with universal 16S rRNA primers,electrophoresis • Week 7 - DNA sequencing reactions, sequencing gels • Week 8 - Read, analyze sequence data

Primer Design Strategy

• Sequences are highly conserved among prokaryotes.

• Segments of amplified fragment show significant variation.

• Fragment size between 350 and 600 bp is optimal for PCR and sequencing from both ends.

–

Primer A - 28 nts - coding strand (~310 - ~340) 5’-CGGCCCAGACTCCTACGGGAGGCAGCA- 3’

–

Primer B - 26 nts - non-coding strand (~770 - ~740) 5’-GCGTGGACTACCAGGGTATCTAATCC- 3’ ribosomal RNA gene (~1450 bp) PCR product (~460 bp) A B

Microbiology Bioinformatics

• Read sequence data from gel • Search database to identify organisms • Retrieve unknown organism’s rRNA sequence, isolate PCR product sequence.

Microbiology Bioinformatics

• Align rRNA sequences corresponding to amplified fragment from diverse organisms.

• Identify specific sequences common to particular groups • Construct phylogenetic tree

Summary

• Bioinformatics exercises… – Are tightly coordinated with laboratory projects – correlate with and support material covered in the classroom – build on earlier exercises – increase in sophistication as students progress through the curriculum – teach students how to work with real research-quality information and tools

Visit the Project Web Page at http://www.lycoming.edu/~newman/models.html