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Archaea Survey

Experiment:

Determining the genetic diversity of archaea in deep-sea hydrothermal vent environments (Edmond Vent System) such as effluent vent water and chimney structures by comparing the archaeal populations of this vent system with those of other high-temperature ecosystems and examining the genetic diversity and phylogenetic organization.


Justification:

  • To possibly discover types of unknown and uncharacterized members of archaea, which will expand scientific knowledge, perhaps serving as an evolutionary link.

  • Discover major habitats/sources of novel deep-sea thermophilic archaea.

  • Future: undertake molecular phylogenetic surveys of the archaeal populations in such environments.


Procedure:

The methods of small subunit rRNA gene sequencing, the cultivation-isolation technique, and the molecular phylogenetic approach will be utilized

  • take samples from various hydothermal vent environments (water and sediment)

  • immediately cool water on ice and store at 4 degrees C, and freeze sediment samples at –85 degrees C until processed

  • extract nucleic acids by physical disruption and chemical lyses of the cells according to the method of Takai and Sako

  • filter vent water through 10-?m-pore size Nitex filters and 6.0-?m-pore-size filter paper, collect microbial particles from these filtered water samples on 0.22?m-pore-size, 47-mm-diameter cellulose acetate filters, then wash filters twice with NET buffer and store at –20 degrees C. (repeat process for samples after crushing)

  • fix vent water and chimney samples filtered through 6.0-?m-pore-size filter paper in 3.7% formaldehyde, and observe under Leica DMRB microscope or Leica MPS 30 camera system

  • amplify small subunit ribosomal RNA genes using LA Taq polymerase with GC buffer perform thermal cycling using the GeneAmp PCR system

  • pool amplified rDNAs from five separate reactions

  • after centrifugation, DNA pellets are resuspended in sterile distilled water.

  • Clone purified rDNAs in vector pCR2.1 using original TA cloning kit, and then build archaeal primer

  • Analyze partial rDNA sequences using SIMILARITY_RANK and ALIGN_SEQUENCE from the Ribosomal Database Project and the gapped-BLAST search algorithm to estimate the degree of similarity to other rDNA sequences.



Sources

Nucleic Acids Research, 2000, vol. 28, No.3, “Structural analysis of DNA sequence: evidenct for lateral gene transfer in Thermotoga maritima”

Applied and Environmental Microbiology, Aug. 1999, “Abundance and Diversity of Archaea in Heavy-Metal-Contaimated Soils”

Genetics Society of America, 1999, “Archaeal DNA Replication: Identifying the Pieces to Solve a Puzzle”

Biochemistry, Vol. 95, December 1998, “Temperature, template topology, and factor requirements of archaeal transcription”

Genetics Society of America, 1999, “Genetic Diversity of Archaea in Deep-Sea Hydrothermal Vent Environments”

Deming, Stanley, N. Experimental design: a chemometric approach. Elsevier Science Publishers, 1944.

Rodriguez-Valera, Francisco. General and Applied Aspects of Halophilic Microorganisms. Plenum Press, 1989.

Primrose, S.B. Principles of Genome Analysis, a guide to mapping and sequencing DNA from different organisms. Blackwell Science, 1995.