Transcript Biotechnology-2

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 - Comparison of mouse clotting factor IX
gene and cDNA.
• 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)
A
PCR product
(~460 bp)
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