the undergraduate genomic sequencing project

11
THE UNDERGRADUATE GENOMIC SEQUENCING PROJECT Zachary Bendiks

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Zachary Bendiks. The Undergraduate Genomic Sequencing Project. Jonathan Eisen. UC Davis Genome Center Lab focus: “ Our work focuses on genomic basis for the origin of novelty in microorganisms (how new processes and functions evolve ).” - PowerPoint PPT Presentation

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Page 1: The Undergraduate Genomic Sequencing Project

THE UNDERGRADUATE

GENOMIC SEQUENCING

PROJECT

Zachary Bendiks

Page 2: The Undergraduate Genomic Sequencing Project

Jonathan Eisen UC Davis Genome Center Lab focus: “Our work focuses

on genomic basis for the origin of novelty in microorganisms (how new processes and functions evolve).”

Metagenomics – assessing the microbial makeup of environmental samples

Bioinformatics - applying computer technology to phylogeny reconstruction

http://www.ucdmc.ucdavis.edu/medmicro/jeisen.html

Page 3: The Undergraduate Genomic Sequencing Project

16S rDNA Present in all prokaryotes. The 16S gene codes for the small rRNA

subunit. ~1.5 kb in length rRNA can be phylogenetically significant

crucial for protein synthesisConserved structure and function across all

organismsUniversal in cellular organismsDiffering rates of evolution throughout the gene can

help identify species Carl Woese (1970)

Page 4: The Undergraduate Genomic Sequencing Project

The Undergraduate Genomic Sequencing Project First attempt by Eisen lab to incorporate

undergraduates Attempts to strengthen mechanical lab

skills by taking undergraduates through the process of isolating genomic DNA.

Learn simple genetic analysis techniques and methods used to identify organisms

Page 5: The Undergraduate Genomic Sequencing Project

Culturing Bacteria Place environmental

sample in liquid overnight culture

Grow liquid culture on nutrient plate

Use dilution streaking to isolate individual colonies, and thus, individual species

http://www.scienceprofonline.org/science-image-libr/sci-image-libr-bacterial-growth-media.html

Page 6: The Undergraduate Genomic Sequencing Project

Genomic Preparations Extraction of genomic

DNA from the cell Lysis of the membrane Cellular proteins and

other impurities are dissolved

Genomic DNA is suspended in rehydration solution

Presence of DNA is confirmed on agarose gel

http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/dna_lab/images/dna_precipitate/174993-1-eng-AU/dna_precipitate_large.jpg

Page 7: The Undergraduate Genomic Sequencing Project

16S PCR DNA polymerase, forward

and reverse primers, and buffers are combined with the DNA

Solution heated through cycles for several hours. Taq polymerase -

thermophilic Allows polymerase to

repeatedly amplify the regions that the primers specify

Confirmed on agarose gel

http://upload.wikimedia.org/wikipedia/en/a/a9/Amit_Yadav_16S_PCR_Phytoplasma.JPG

Page 8: The Undergraduate Genomic Sequencing Project

PCR Purification Free-floating primers,

buffers, and other impurities are still in the solution. Need pure DNA for sequencing

Use a series of solutions and a filtering tube to remove contaminants

Pure DNA solution can then be diluted to the proper concentration and sent for sequencing

http://www.favorgen.com/jpg/FAEPK-02.jpg

Page 9: The Undergraduate Genomic Sequencing Project

Genetic Analysis Geneious – creates alignments between

fragments and generates consensus sequence

BLAST – compare sequence to a database of organisms

GOLD – lists the number of in progress or future projects for organisms

Page 10: The Undergraduate Genomic Sequencing Project

http://www.geneious.com/geneious_theme-theme/images/biomatters/screenshots/3.7_sequence_organize_sequence_visualize.png

Page 11: The Undergraduate Genomic Sequencing Project

Looking Forward My sequenced organisms so far

Staphylococcus pasteuriEnterobacter ?Bacillus amyloliquefaciens6 more currently in sequencing facility

Undergraduates will be assigned an organism and write a summary report of it and publish the full genome to a database