Transcript Michael Clement Dr. Stephen Giovononi

Marine Bacteria Cultivation
Michael Clement
Dr. Stephen Giovannoni
Purpose
• To culture and grow ecologically important
marine bacteria from a complex marine
environment
• Cultivation of marine microbes is difficult
but necessary for phenotypic and
genotypic analysis
Background
• We know lots of different marine
organisms exist because we can detect
their DNA
• Most organisms we can detect we cannot
grow in the lab
Background
• SAR11 is abundant in marine waters
worldwide
• SAR11 plays a large, but not well
understood role in the carbon cycle
• SAR11 has been cultivated in the
laboratory
Growth Medium
• Oregon coast seawater filtered with a 0.1
micron filter
• Autoclaving vs. Not autoclaving
Inoculation
• 24-well Teflon plates
• 5ml medium/well
• Inoculate at 2.5
•
•
•
cells/well
Inoculated 144 wells
24 negative control
wells
Grew for three months
Growth Assessment
• Guava flow cytometer
• DNA binding dye
• Assess cell density of
culture
Growth Assessment
• Positive wells determined by visual
comparison of Guava reads to
uninoculated wells
Analysis
•
•
•
•
PCR
Gel electrophoresis
RFLP
Determine unique
patterns
Growth results
• 34% culturability
• Some co-cultures and some pure culltures
• Negative control wells had no growth
RFLP Results
>7C6R_8294-27F_H03_009.ab1 Uncultured Thiotrichales
ACTTAACGCGTTAGCTTCGCCACTAAAGGGTAAATCCCCCCAACGGCT
AGTTATCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTT
GCTACCCACGCTTTCGTACCTCAGCGTCAGTATTGGTCCAGAAAGCTG
CCTTCGCCATTGATGTTCCTTCTGATATCTACGCATTTCACCGCTACAC
CAGAAATTCCACTTTCCTCTACCATACTCTAGTTGACCAGTTTCAAATG
CAGTTCCCAGGTTAAGCCCGGGGCTTTCACATCTGACTTAATAAACCG
CCTACGCACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTC
CGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTAA
AGTTAACGTCAAGGCTAACGGTTATTAACCGCTAACTTTTCTTCACAAT
TGAAAGTGCTTTACAACCCTCAGGCCTTCTTCACACACGCGGTATTGC
TGGATCAGGGTTGCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCC
GTAGGAGTTCGGGCCGTGTCTCAGTCCCGATGTGGCTGATCATCCTC
TCAGACCAGCTAAAGATCGTCGCCTTGGTAGGCTTTTACCCTACCAAC
AAGCTAATCTTACGCAGGCTCATCTGATAGCGTGAGGCTCGAAAGTCC
CCCACTTTACTACGAATAGATTATGCGGTATTAATCCGAATTTCTTCGG
GCTATCCCCCACTATCAGGCAGATTCCTACGCGTTACTCACCCGTCCG
CCACTCGACGCCTACTAGCAAGCTAGTATCGTTTCCGTTCGACTTGCA
TGTGTTAAGCATACCGCCAGCGTTCAATCTGAGCCAT
2 Uncultured
Alphaproteobacteria
13
10
1
1 Thiotrichales
2 plastid/
1 Uncultured
Gammaproteobacteria
cyanobacteria
Future Application
• Two mixed cultures are undergoing
continuing work
– Uncultured Gammaproteobacteria (SAR 86?)
– Plastid or cyanobacteria
• Frozen stocks of cultures
Conclusion
• Ongoing focus of the Giovannoni lab is to
cultivate new species as well as diverse
members of already cultivated species
• Filtering medium verses autoclaving it is a
potentially viable means of cultivating rare
or uncultured organisms
Special thanks to…
• Dr. Stephen Giovannoni
•
•
•
For taking me into his lab
Dr. Kevin Ahern
For his wonderful organization of this program
Paul Carini
For all of his help day in and day out
The Howard Hughes Medical Institute
For their continued support of this program