Part Two

Identification of bacteria by DNA sequencing

Beckman CEQ 8000

Capillary Head Sequencer

Sequencing Reaction: One Primer, labeled dNTPs, other PCR reagents

Sequences emailed from facility.

FinchTVTM: A free Chromatograph Viewing Program

Each sample: Analyze overall quality of sequence

Example Chromatogram:

Identify Unknown Bacteria at NCBI:

Basic Local Alignment Search Tool

NCBI: A Worldwide Resource

Origination of some “HITS” to our sequences

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

RESULTS: Monday 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

RESULTS: Tuesday Lab 2005

RESULTS: Alignment All Samples Monday Lab

Conserved and Variable Regions

Generated using ClustalW(free Alignment maker)

Unrooted tree (generated by Phylip's Drawtree: another online program)

Alignments are the Basis of Phylogenic Trees

16SrRNA DNA amplified using Primer Set #1

RESULTS: Relatedness

Example Next Slide: Pseudomonas aeruginosa

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.

Full length E. coli 16S rRNA gene

Region of Alignment PCR sample

Pseudomonas aeruginosa isolate: 82% identity match with E. coli

Primer Set #1

~425 bases of alignable DNA sequence generated

Full length E. coli 16S rRNA gene

Pseudomonas aeruginosa isolate: 83% identity match with E. coli

Primer Set #2

Region of Alignment of PCR

~280 bases of alignable DNA sequence generated

Class Results on Spreadsheet

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.

1) Peer Reviewed Journal Articles

2) Conferences: a) Short Presentations b) Poster Sessions

3) Informal Networking, Retreats etc.

Real Deal

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 differentlyPOSITIVE

Future directions………….Applications

Attention grabber

Compare to those we know

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

2a) Science Talks

2b) Poster sessions: Adult versions of the Science Fair

Poster session in the parking structure!

Sections for the Poster

TITLE(Name; Organization)

INTRODUCTION

METHODS

RESULTS Figures and Tables are good….Use some we made andMake some of your own

Gels, Chromatographs, Alignments etc…

Goal: to show a story start to finish in a visual way

CONCLUSIONS / Future Research

Making a Poster

STANDARD WAY: A Giant Power Point Slide

Power Point: under File MenuChoose “Page Set Up”Select Dimensions for PosterStandard 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

Often Easiest to:

Put together text in Word or Regular Power Point size slides

Cut and paste into the giant slide.

Groups for Posters…..

End of semester poster setup and presentations

Check talk beforehand

Please practice with your group so everyone knows their part

Want to Post Your Sequence?

http://www.ncbi.nih.gov/Genbank/index.html

END