Part Two

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Part Two

• Identification of bacteria by DNA sequencing

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Sequencing Reaction One Primer, labeled dNTPs,
other PCR reagents
Beckman CEQ 8000 Capillary Head Sequencer
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Sequences emailed from facility.
FinchTVTM A free Chromatograph Viewing Program
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Each sample Analyze overall quality of sequence
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Example Chromatogram
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Identify Unknown Bacteria at NCBI Basic Local
Alignment Search Tool
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NCBI A Worldwide Resource
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Origination of some HITS to our sequences
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RESULTS Monday Lab 2005
Student Bacteria Source Primers NCBI NCBI match
Monday BH Control Set One Bacillus subtilis 98
Monday PM Control Set One Pseudomonas aeroginosa 98
Monday PS Control Set One Citrobacter freundii 99
Monday KY Control Set One Citrobacter freundii 95
Monday MW Control Set One Enterococcus faecalis 99
Monday MT Control Set One Enterococcus faecalis 98
Monday ZZ Soda Can Set One Psuedomonas geroginosa 99
Monday SN Soda Can Set One Neisserra cinerea 98
Monday DR Soda Can Set One Brevundimonas vesicularis 99
Monday JC Soda Can Set One Serratia marcescens 99
Monday AE Soda Can Set One Klebsiella sp 94
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RESULTS Tuesday Lab 2005
Student Bacteria Source Primers NCBI NCBI match
Tuesday TMM Control Set Two Pseudomonas aeruginosa 96
Tuesday JM Control Set Two Pseudomonas aeruginosa 96
Tuesday ES Control Set Two Enterococcus faecalis 99
Tuesday AM Soda Can Set Two Brevundimonas vesicularis 99
Tuesday MO Soda Can Set Two Neisseria subflava 99
Tuesday DA Soda Can Set Two Klebsiella planticola 98
Tuesday BB Soda Can Set Two Klebsiella sp. 98
Tuesday BK Soda Can Set Two Neisseria sp. 95
Tuesday AP Soda Can Set Two Neisseria sp. 99
Tuesday JS Soda Can Set Two Klebsiella sp. 98
Tuesday IF Soda Can Set Two Nesseria sp. 97
Tuesday JW Soda Can Set Two Neisseria sp. 99
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RESULTS Alignment All Samples Monday Lab
Conserved and Variable Regions
Generated using ClustalW (free Alignment maker)
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RESULTS Relatedness
Alignments are the Basis of Phylogenic Trees
16SrRNA DNA amplified using Primer Set 1
Unrooted tree (generated by Phylip's Drawtree
another online program)
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RESULTS Generating a longer fragment
The first primer set amplifies the middle of the
16s DNA, and the second primer set amplifies an
end. The sequences overlap so in the future these
primers could be used to get a longer sequence.
Example Next Slide Pseudomonas aeruginosa
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Primer Set 1
Pseudomonas aeruginosa isolate 82 identity
match with E. coli
Region of Alignment PCR sample
Full length E. coli 16S rRNA gene
425 bases of alignable DNA sequence generated
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Primer Set 2
Pseudomonas aeruginosa isolate 83 identity
match with E. coli
Region of Alignment of PCR
Full length E. coli 16S rRNA gene
280 bases of alignable DNA sequence generated
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Class Results on Spreadsheet
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Communicating Science The Real WorldIt is just
as important to be able to communicate your
results to others as it is to gather and
interpret data.

• Peer Reviewed Journal Articles
• Conferences
• Short Presentations
• Poster Sessions
• Informal Networking, Retreats etc.

Real Deal
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2) Talks in General Title Often good to come
up with your own Background What was the
question they were trying to answer? Methods
Results Figures are often the best way to
show results Conclusions.. Critiques. What
you would do differently POSITIVE Future
directions.Applications
Attention grabber
Compare to those we know
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2a) Sample Science Talk One variant of an
Intro to Our Project Methods Some Key
Results.not all
Attention grabber
Are the Bacteria Dangerous?
Does this change your view?
Discussion Critique Put in Posters
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2a) Science Talks
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2b) Poster sessions Adult versions of the
Science Fair
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Poster session in the parking structure!
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Sections for the Poster TITLE(Name
Organization) INTRODUCTION METHODS RESULTS
Figures and Tables are good. Use some we made
and Make some of your own Gels,
Chromatographs, Alignments etc Goal to show a
story start to finish in a visual
way CONCLUSIONS / Future Research
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Making a Poster STANDARD WAY A Giant Power
Point Slide Power Point under File
Menu Choose Page Set Up Select Dimensions
for Poster Standard Poster Sizes Width 48
inches Height 36 inches
Slides landscape Or Use a Template (steal
from a friend) Google poster template science
or use link on webpage
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Often Easiest to Put together text in Word or
Regular Power Point size slides Cut and paste
into the giant slide. Groups for Posters..
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End of semester poster setup and
presentations Check talk beforehand Please
practice with your group so everyone knows their
part
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Want to Post Your Sequence?
http//www.ncbi.nih.gov/Genbank/index.html
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END