Heterotrophic Bacterial Communities Associated with Desert Gypsum Rock

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Heterotrophic Bacterial Communities Associated
with Desert Gypsum Rock

• By Leigh Ann Beasley
• (Research Supervision by Dr. Furlong)

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Background Information

• Gypsum rock
• a translucent material
• found in deserts
• microbial communities
• Cyanobacteria
• Heterotrophic bacteria
• play important roles in the desert ecosystem
• weather the gypsum rock
• prevent erosion

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Gypsum Rock
geology.kangwon.ac.kr/ lecture/rock/sed/gypsum.jpg

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Goals of The Research

• Extract the DNA from microbial communities
• PCR amplifiy 16s rDNA from heterotrophic bacteria
• Build a 16s rDNA clone library of the
  heterotrophic bacteria found on the gypsum rock
• Analyze the 16s rDNA gene to determine
  heterotrophic community sturcture

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Revised Goals of The Research

• Culture the heterotrophic community of the gypsum
  rock
• PCR amplifiy 16s rDNA from heterotrophic
  community
• Build a 16s rDNA clone library of the
  heterotrophic bacteria found on the gypsum rock
• Analyze the 16s rDNA gene to determine
  heterotrophic community sturcture

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Culturing Method

• Series dilution (a total of 10 samples ?120
  plates)
• DNB-CaSO4 media

0.1 ml
1.0 ml
1.0 ml
1.0 ml
1.0 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
1 ml physiological saline
10 mg gypsum
3 Xs
3 Xs
3 Xs
3 Xs
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DNA Extraction From Cultured Heterotrophic Plates

• 2 possible strategies
• Collect DNA from isolates
• Collect DNA from all colonies
• Collected ALL colonies from non-contaminated
  plates (42 plates total)
• Extracted using Ultra Clean Soil DNA Isolation
  Sample Kit by Mo Bio
• The Results..

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DNA!!!
We determined that there was 20 ng DNA per ml
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Amplifying the 16s rDNA Gene

• PCR
• Ran 2 PCR samples
• 2.5 ml of extracted DNA
• 1 ml 27F primer
• 1 ml 1492R primer
• 20.5 ml sterile HPLC water

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Cloning

• Followed the Invitrogen protocol for TOPO TA
  cloning
• LB/Amp/X-gal plates
• Amp/Kan selection
• Select for clones with plasmid

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Screening

• Used the Blue/White method
• Used b-galactosidase assay
• Screened for transformants with insert
• selected white colonies
• Streaked the colonies onto grid plates (LB media
  with Kanamycin added)
• 26 colonies per plate 13 plates total

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Sequencing

• Packaged the clones
• 96-well culture block
• Freezing media
• Used well-defined isolates
• Sent to SeqWright
• Unfortunately some of the sequences were unusable
  (cross contamination?)
• 34/98 sequences received were used

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Firmicutes
Euryarchaeota
Alpha Proteobacteria
.031 evolutionary distance
Beta Proteobacteria
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Future Research Plans.

• Culturing method for cyanobacteria
• Overshot dilutions
• Extract DNA directly from gypsum sample
• CaSO4 material
• Method for extracting DNA from bone

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Acknowledgments

• Dr. Furlong
• Dr. Jordan
• Dr. Braun
• Dr. Clower
• Dr. Whittman
• Chris Lasher

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Questions?
?