Geochemistry & Ecology of Red Mat Systems (GERMS)

Undergraduate Summer Research Program

 

Week Five

DNA Sequence Analysis

 

Sequencing Reaction Set – Up

 

Procedure:

Each of the five samples are separated into four tubes.  Each tube is label T, G, A, or C.  Stop nucleotides are added to the corresponding tubes.   When run on the gel, the samples will have a one base difference from one of the other four lanes.  This is what allows us to determine the sepquence of the gene. 

Jana’s Sequencing reaction set-up

 

Preparing Gels:

 

Procedure:

Liz is pouring the buffer into the lower well of the gel.  There is an upper well also that holds buffer.  The wells are attached and filled after the gel is secured in the machine. 

Pouring buffer into the lower well

 

 

 

 

 

 

 

 

 

 

Reaction Finish and Load:

 

Procedure:

I’m carefully loading my samples onto the gel with a special pipette tip that is flat and slips between the teeth of the comb. 

LOADING SAMPLES ONTO GEL

LOADED GEL AND SET-UP

 

 

Reading, Editing and BLASTing Sequences

 

SEQUENCE INFORMATION

 

Clone Number Analyzed:

 

Edited Sequence:

 

 

 

Fairy Green 21

tGAgcttgtaTcGcTccctttGGgcGattgGGccctaGAtgAtgcTGagcGGCGCAGtgT

GATGGAaaTTGcAGAATtGCCTTgGtTGAGCGGCCGaCCggGTTacCcTgtTACGTCcTT

AcCCCAGTaAttAgTCCCttCCCTtGtCGttCaTCccCattaGgTttGTGtagTtACGTc

CTtGgGaGTgGaCAACctCCAatgtgTgaCGGGCGGtgTgTaCAAGgaCCggGAACGtaT

TTaaCGaAGTATgtTgTACCctaGAtTacttgCtgAaTTccTCcTcAGTAGGCGATGTTT

CAGCCcTtAAAtTtTtACCTAGtcCACGTtTTaTgTGgATTtgtcCTTTtTTCGcGaGCT

TtCAatCCCTTTtTTtTTaGCAtTTTTATgTACGtGTTTAGCCCAtTTcGTAAGtGGGCA

CgCcT

 

Fairy Green 22

agaAAAAAaaCGaCCAcTaTaGggCGAAtTGGGCCCTCTAGATGCATGCTCGaGCGGTCG

TCAGTGTGATGGATAtCTGCAGAATgCGTCCTTGCGGTtCCTcGGTCGTTgcAGaGTTTG

AACCTGGTtCAGgTTGAACGTTGgCGtTcTtTTTAACACAtTCAaGTTGAACGaAGGCTT

TCGGTCACTtAGTGGTcGGaCTTGTGAtTAACGCGGAgTaATCTGCCCcgAGTGACGGtT

ACAACATcTGGAAAtgGCTGCtAATACCTTGATGTGCCGTAGAGGgGAaAtATTTTTATG

GtCCtaGGaTGAGCCTTGTGTCTGaTtAGCAGTTGtTGGGGTAAGAGCCTACCAaTgTGA

CGaTCAGgAGCTTTTcTGAGtTGtTGTTCAGCCACATTgTGACTGTttCAcGTcCCAGAc

GTcTACGgGTGGCAGtAGTGgTtTATTTgCtGtAAGGgGCGTaATCTTTacGttgCAAGg

TC

Fairy Green 23

CTCTtgATACaTaCTCAACcCCCcCCAATATAATAAATATCTAcAAAATTCAAcCTTAAa

TCATACAACCtACcAAaATTACCTTGTTAcGACTTcACCCCAATAATaatCcCTAacTTa

atAATAcTGCtGatTtGtGTTAAAATaACAAaTtaAaAAATgAAcAAATAACATGAtATA

AaaAAaAAAtGGTAtTaCAAGAAcCAGAaAAatattAaaaAAAAGatACtAAGCtaAAAt

tGAtGAtAAtt

Fairy Green 24

gGcCTcTAGATGCATGCTTGAGTGGTcGTCAGTGTGATGGATATcTGCAGAATTCGCCCT

TGGCTCGAGCGGCCGCGGaTCCgcGGCTGTCAGAGTTTGATCCTGGCTCAGGATGAACGC

TGGCGGCGTGCTTAACACATGCAAGTCGAACGGGAtCTTCGGATCTAGTGGCGGACGGGt

GAGTAACACGTAGGAATcTACCCACAGGAGGGGGACAACAGTTGGAAACGACTGCTAATA

CCCCATAGGGCGCGaGCTGAAAAGAATTtTcGcCTGTGGATGAGCCTGCGTgCTGATtTA

GCTAGTTGGtGGGGTAATGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGA

TCAGCCACACTgGGACTgAGACAcGGGCCAGACTCCTACGGGGAGGGcAGCAGTGGGGAA

TTTTCCGCAATGGGGCGAAAGCTGgACGGAGCAACGCCGCGTGAGGGAGGAAGgCCCtTg

ggtTGtAAACC

Fairy Green 25

gGCCCTCTAGATGCATGCTCGAGCGGcCGCCAGTGTGATGGATATCTGCAGAATTCGCCC

TTGCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGTCTG

CTTAACACATGCAAGTCGAACGGAGTGcTTCGGCACTTAGTGGCGGACGGGTGAGTAACG

CGTGAGAATCTGCCCTTAGGACGGGGACAACAGCTGGAAACGGCTGCTAATACCCGATGT

GCCGAGAGGTGAAACATTTTATGGCCTGAGGATGAGCTCGCGTCTGATTAGCTAGTTAGT

GGGGTAAGAGCCtACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGCCACAC

TGGGACTGAGACAcGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGaATTTTcCgCAATG

GGCGCAAGCttGACGGAGCAAGACCGcGTGCGGGAGGACGGCCTATTGgtTgtAAACCGC

TTTTGATAGGGAAGAAGTACTGaCGGTACCTATCGAATCAGCATCGGCTAACTgCGTGCC

AGCAGcCgCGGTAATACGgAGgaTG

 

 

 

 

 

 

 

 

 

 

 

 

 

TOP FIVE DIFFERENT BLAST HITS

Be sure to use different hits (uncultured = same;  go down until you find specific species, including cultured isolates)

 

For source, I am looking for where it came from (e.g. termite gut);  if none is listed, read the title reference and abstract (if published) to glean this information.

 

 

 

 

 

Fairy Green 21

Top Accession #

Organism Name

Source

 
AF176296
 
Pasteurella multocida
 
Bacterial phylogenetic clusters revealed by genome structure

AF320982

 

Desulfitobacterium frappieri

 
A new ecological niche for the members of the genus
Desulfitobacterium and a description of Desulfitobacterium
metallireducens sp. nov., an Anaerobic Bacterium that Couples Growth to the Reduction of Metals, Humics, and Chlorinated
Compounds

Y15855

 

Moraxella osloensis

 
sludge community

AJ278106

Klebsiella sp. RS1

 
Bacteria from the rhizosphere of rice

AF533685

 

Sinorhizobium meliloti (Rhizobium meliloti)

Phylogenetics of rhizobia isolated from indigenous legumes from Northwest of Western Australia

 

 

Fairy Green 22

Top Accession #

Organism Name

Source

 
AJ431219

 

proteobacterium BHI60-11

 
Novel bacterial lineages from enrichment cultures with Alvinella pompejana white tubes collected on East Pacific Rise at 13oN

AB069653

 
Bradyrhizobium sp. PAC50
 
Two types of symbiotic root nodule bacteria isolated from yam bean

 

No other matches

 

 

 

Fairy Green 23

Top Accession #

Organism Name

Source

AB107162

Firmicutes bacterium K-2063

 
Characterization of Sphingomonas spp. found as predominant members in the culturable bacterial community of a green pigment-containing sclerotium grain from Mt. Myko (Japan) volcanic ash soil

 

AB021188

Bacillus halophilus

unknown

AJ006853

sulfate-reducing bacterium mXyS1

unknown

AB030223

Catenibacterium mitsuokai

 
a gram-positive anaerobic bacterium isolated from human feces

AF526913

Bacillus odysseyi

 
Microbial characterization of the Mars Odyssey spacecraft and its encapsulation facility

 

 

 

 

 

 

Fairy Green 24

Top Accession #

Organism Name

Source

AF001480

 

Synechococcus sp. WH 8101

Rapid diversification of marine picophytoplankton with dissimilar light-harvesting structures inferred from sequences of

Prochlorococcus and Synechococcus (Cyanobacteria)

 

 
AF132791

 

 
Gloeobacter violaceus PCC 8105
 
Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis

AE017162 AE017126

Prochlorococcus marinus

 
Genome sequence of the cyanobacterium Prochlorococcus marinus

AJ289785

cyanobacterium 5X15

 

Molecular and pigment studies of the picophytoplankton in a region

            of the Southern Ocean (47 to 54oS,141 to 144oE) in March 1998

 

No other matches

 

 

 

Fairy Green 25

Top Accession #

Organism Name

Source

 
AF263344

 

Oscillatoria sp. J-24-Osc

 
cultivated Antarctic oscillatorians(Cyanobacteria)

 

AF091110

 
Plectonema sp. F3

 

oxygenic photosynthetic bacteria

AF091150

 

Anabaena cylindrica

Anabaena (cyanobacteria)

AY615503

 

Oscillatoria spongeliae

dictyoceratid sponges

AF067818

Cylindrospermopsis raciborskii

Two morphological forms of Cylindrospermopsis raciborskii

            (cyanobacteria) isolated from Solomon Dam, Palm Island, Queensland

 

 

 

Discussion:  (Hint: Comment on the BLAST output in terms of what kinds of research you saw going on out there:  variety of research sites?  Ball-park proportion of data that represents "uncultured" retrieval that use DNA-only information?)

 

For FG-22, mostly cloning vector matches came up.  This might be due to a poor gel run, or the Ecoli transforming vectors with inserts.  That is why there are only two entries above for FG-22.  For FG-24, the whole result list a repetitive list of the same organisms, and uncultured organisms.  That is why there are only four entries above for FG-24.  I found a lot of research is being done on pathogenic organisms, and cyanobacteria.