The Scientific Method Assessing the metabolic potential of Caltechs - - PowerPoint PPT Presentation

The Scientific Method Assessing the metabolic potential of Caltech’s microbial community… part I

Metagenomic and functional analysis of hindgut microbiota

• f a wood-feeding

higher termite

The Terms of Metagenomics

Metagenomes – genetic material recovered directly from environmental samples Metagenomics – the study of metagenomes

• Clone specific genes (16S rRNA gene) to produce a

profile of diversity in a natural sample

• Sequence to get largely unbiased samples of all

genes from all the members of the sampled communities

• Use bioinformatics to analyze the data

The application of computer science to the field of biology

What is Bioinformatics?

The Algorithms

Phrap – used for DNA sequence assembly FgenesB – ab initio gene prediction engine BLAST – finds regions of local similarities between sequences and aligns them Pfam – collection of protein families and analysis tools PHYLIP – infers evolutionary trees (phylogenies)

Pfam Demo

http://pfam.sanger.ac.uk/

MAHGCSGGAMSRFVFLGVALALLGGATSPAAAAPRVTPVVVDVDDYGADPTGRTDSTPAVAAALRHAKSVDRPVRIVF SKGTYQLYPERAETRELYMSNTVGADQRYRDKKIGLLVEDMHDVTVDGGGAKLVHHGLQTAFASIRSTDVTFQNFSFDYA APEVIDATVATTGVTDGHAYRVLKIPAGSPYRVNGTHITWLGETSPATGQPYWSGVDGLQYTQIHDPEAQRTWRGDNPL FNDVAAVTDLGGRRIRIDYTTAARPADAGLVYQMRLIERTEPGAFIWESKNVTMRSMNAYYLQSFGVVGQFSENISIDKV NFAPDPRSGRSTASFADFVQMSGVKGKVSITRSLFDGPHDDPINIHGTYLEVVGKPGPSTLTLAYKHPQTAGFPQFAPGD EVEFATKRTMTPLADAHAQVTAVDGPSGMDHTKPLTTMTVTFDRPVPAGVETGGTVVENITATPSVVISGNVFRNVPTR GILVTTRKPVLITGNRFDGMSMASIYVSADAYQWYESGPVADLTIRGNSFTRPSGPVIFVEPTNQVIDPATPVHHNISVEH NSFDIGDVTVVNAKSVGGFAFT GNTVRRLDGADHPPYTSPLFVFHGSSGIRIARNHYDKGLNTSVVTD

The Scientific Method

A body of techniques for investigating phenomena, acquiring new knowledge, or correcting and integrating previous knowledge Four essential elements:

• 1. Characterizations –observations, definitions of

subject of inquiry

• 2. Hypotheses—theoretical, hypothetical

explanations

• 3. Predictions—reasoning
• 4. Experiments—testing all of the above

The Volta Experiment

• 1. Characterizations of flaming pond
• 2. Hypotheses
• 3. Predictions
• 4. Experiments

Ponds produce methane gas Methanogenisis

• f life in the

pond created the gas

Microorganisms living in the pond may produce methane during metabolism

Isolate and identify

• rganisms based
• n what they

metabolize Interpret data and draw conclusions

Metabolic potential of Caltech’s ponds

Mini Lab

Collect sample from a pond and plate it Monitor the plates to track colony growth Determine most diverse plate and collect another sample from that location Metabolic assay of the sample and isolates from the plate

The section that locates the most metabolically active location on campus will receive a fabulous prize!!!

BBB Pond Baxter Pond Turtle Ponds

Sample the environment! Week 4

• Isolate a sample of pond water for a

representative microbial population

– Baxter, BBB, turtle ponds

• Plate serial dilutions pond water on 1% TSB agar

plates

– Trypticase Soy Broth (TSB)

• Incubate plates to allow colonies to grow

– One week of growth time at room temperature – Check on their growth during the week

Biolog Metabolic Assay Week 5

• Different populations of microbes may utilize

different carbon sources

• Identifying which carbon sources can be used can

allow for identification

• 96-well plate format allows for the simultaneous

testing of 95 different carbon sources at one time

• If cells proliferate on a certain well, they are able

to metabolize the carbon source

100 10-1 10-2 10-3 100 10-1 10-2 10-3

Week 4: Week 5:

Incubate for one week at room temperature Baxter BBB Turtle pond Individual isolates As a section, choose which location you think has the greatest metabolic potential Indicator dye

Week 6:

Click on Sequence Search

• 1. Paste your

sequence in the box

• 2. Click ‘Go’

Click on the family name you want

Click on Species

Click on Tree Tab

Use the species, sequences, and regions numbers to interpret tree