Geochemistry & Ecology of Red Mat Systems (GERMS) Undergraduate Summer Research Program Week Five – DNA Sequence Analysis Imperial Runoff-Green Elizabeth Jacobsmuhlen

Reaction Set-Up:

Procedure: This is a picture of the clones to be used for DNA sequence analysis. These clones were: I6, I7, I8, I9, and I16. They were chosen based on the plasmid isolation gel from last week. This gel supported that of the 20 clones ran on the gel, these five clones contained both insert and vector.
Procedure: The clones are being placed in the PCR machine as a warming up cycle so that they could then be prepared further and then loaded onto the DNA sequencing gel.

Preparing Gels:

Procedure: The picture to the right shows the glass plates being thoroughly cleaned with double distilled water and then 95% ethanol to dry/buff the face of both plates. Due to the thin nature of the gel a small imperfection on the glass plates when the gel is running, such as a dust particle, could cause the gel to run improperly.
Procedure: After the faces were cleaned and placed face-to-face, the outside of the glass was also cleaned, to make sure that there was nothing on the glass that would interfere with the lazar reading the gel.
Procedure: Before the gel can be ran the machine must be calibrated and the name of the sequence loaded into the computer.

Reaction Finish and Load:

Procedure: The picture to the right shows the buffer (1X TBE) being poured into the bottom buffer container. Buffer was also added to the top buffer container.
This picture shows the top electrode connection being made.

Reading, Editing and BLASTing Sequences:

SEQUENCE INFORMATION

Clone Number Analyzed: 7-20-04-germs_liz_I_6green_800

Edited Sequence: tcccttcataggtcactatagggcgAAttGGGCCCTcTAGATGcaTGCTCGAGcGGCCGC

CAGTGTGATGGATACTCTgCAGAATTCGCCCTTgGCTCGAGCGGGCCGCCCGgGtTACCTTGTTACGAcTTATCCTGaGCCAGGaTCAAtCTCTGCAGcGGCCGcGGATCCGCAAGGGcGaATTcCAGCAcACTGGCGGCCGgTTACTAGTGGaTCCgaGCTcGgcTACCAtGcTTgGCGTAATCATggTCATTGCTGTTtCCGTGTGAAATTgtTATCCGCTCAcaaTTcCACACAacATACGaGCCgGaaGCATAAAGtGTAAaGCCTGGGGtGCCTaAtgaGtGAGCTAACtCAcAtTAATTgGTTGCGCTtAcTgcCCGTCTtTCCAgTCgggaAaACcTtTTCgTgtcCAgCTcTCATTaaTtgTatTcctgttCcAATCggtGTcGggggcgAGtAGtGCGGgtKCGCTAaTCgcggGAgGTCTCCTGatCGCTtACtCTgTgtActCAA

Top Accession #	Organism Name	Source
AB069651	Rhizobium sp. JEYF14	Symbiotic root nodule bacteria isolated from yam bean

*No more accession numbers were listed because they were all from vector sequences. This could mean that the vector was sequenced instead of the insert or that the insert was bad.

Clone Number Analyzed: 7-20-04-germs_liz_I_7green_800

Edited Sequence: TcgagTtgTaATAcGtCTCACTATAGGcGAATTGGGCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTGGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGaCTTAGTCCCAATCAAGAGTTGAGAGTTAGGCGCTTGCCCCCTTgcGgTTgGCACGgCGACTTcGcTCCCCCCTCTCTtTCGtGGCTTgaCGgGCGGTGTGTACAAGgCTCAGGcaCACAtTCACCGCGGTATTGCTGaCCCgCGaTtACTAGCGaTTCCGaTCTTCATGCAGgCGAGTTgAGCCTaCAcatTGaaaCTggggCGTGCTTTcTTgGgATTcGCGTCCacTCGCGTgGTaggTCCCTTTgTAcaCGtCatTgTaGcACGtcTgaAgCCctggGCATaacagcCCATccGGccTTcAtgtcAGTctCgtcCCttCccgCcCGttcgagcCgCcCGCctCtccccCgaCggtCcCtgCccttaCCGcCgCGCTCcCgGatCTcCCggGGGgGctttggGTCcctGTgCCCccTtgGCCCccgTCttggCccggCCCCTCgCCcGgCggT

Top Accession #	Organism Name	Source
AJ231189	Planctomyces sp. 599	Environment – usually soil or water
X85248	Gemmata obscuriglobus	Planctomycete bacteria from postlarvae of the giant tiger prawn,
AF399914	Pirellula staleyi	freshwater isolate from Campus Lake, Baton Rouge, LA, USA
X62911 S39795	Planctomyces limnophilus	Australian terrestrial environment
AJ390478	uncultured soil bacterium PRR-7	Bulk soil and rice roots of flooded rice microcosms

Clone Number Analyzed: 7-20-04-germs_liz_I_8green_800

Edited Sequence: ccgaatgtaacacGactactatagggcgAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTGGCTCGAGCGGCGCCCGGGTTACCTtgTTACGACTTATCCTGAGCCAGGATCAAACTCTGCAGCGGCCGCGGATCAAGGGCGaATTcCAGcACACTgGCGGCCCGTTACTAGTGGaTCCgaGCTCGGTACCAaGCTTGGCGTatATCATGGTCATAGCTGTTTcCtggTGTGAaATTgTTATCCgtTcAACAaTTTCAtACAaCATACGtGCCCGgaaGCATAAaggTgTAAaGCCctGGgGttgCCcTAatgCAGTgaGCtTAACcTCACAttAAAtTgtgtTggCGTTcACTgcCCgcTtTcCCAgTCgtGgSAaACCcCTCTCtgtgCccAtccTTcTATTcacAcTCcacgCt

Top Accession #	Organism Name	Source
AB069651	Rhizobium sp. JEYF14	Symbiotic root nodule bacteria isolated from yam bean
AB069654	Bradyrhizobium sp. PAC24	Symbiotic root nodule bacteria isolated from yam bean
AJ431219	proteobacterium BHI60-11	Cultures with Alvinella pompejana white tubes collected on East Pacific Rise
AB069653	Bradyrhizobium sp. PAC50	Symbiotic root nodule bacteria isolated from yam bean
AB060974	Rhodococcus opacus	Soil

Clone Number Analyzed: 7-20-04-germs_lIz_I_9green_800

Edited Sequence: ttgaaTtgTAatcGacTActatAGggcGAaTtGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTgCAGAATTCGCCCTTGATCCGCGGCCGCTGCAGAGTTTgATCCTGGCTCAGGACGaACGCTGGCGGCGTGCCTAATGCATGCAAGTCGAACGCACCCGcTTCGGCGGTGAGTGGCGCACGGCTGAGTAACACGtGGGAACCCGcCCCCCaGTGGGGGaTAACGgGaCGaAAGtTgCGcTAATCCCGcATACGtCCTTcGGGGgaAAgcGCA

Top Accession #	Organism Name	Source
AB041226	Roseiflexus castenholzii	Red bacterial mat in the natural environment
AY063760	Eikelboom Type 1851 Ben52	Bulking activated sludge plant
AJ431219	Proteobacterium BHI60-11	Alvinella pompejana white tubes collected on East Pacific Rise
AY395567	Candidatus Chlorothrix halophila	Hypersaline microbial mats
AB064705	Uncultured Bacterium	Microbial community

Clone Number Analyzed: 7-20-04-germs_liz_I_16green_800

Edited Sequence: gcaCCCCcctatgtctactatagggtgAATTGGGCCTcTAGATGCATGcTcGAgcGGCgC

AGCGCGACGGACACCCGcAGAAttgGCCtTGGGTTGAGcGGcCGgcCGGGTTAcCtTGTTAgGActtcACCcAGtCATGAGCCCTGgAtcAGGTGgCcgccTggGcGAACggGTTAGGGCAACAAgTTCGGGgGTGGgCAAaTtcGATGGTGTGAcGGGcGGTGTGTAtAAGGtcCGGGAAcGTATTGAcGGggGTATGcTGAGC

Top Accession #	Organism Name	Source
Z21633	Leptospira biflexa serovar Jequitaia	Does not say, but commonly isolated from water or soil sample contaminated with Leptospira species.
AJ007866	Unidentified Cyanobacterium	Environmental Samples – freshwater source
Z21635	Leptospira noguchii	Does not say, but commonly isolated from water or soil sample contaminated with Leptospira species.
AF132792	Prochlorothrix hollandica	Monophyletic group arising near the root of the cyanobacterial line of descent
Z21638	Leptospira wolbachii	Does not say – but has been isolated from pigs

Discussion:

Much of the research hits generated during the BLASTing process were of rearch concerned with environmental samples some of freshwater sources and root samples. There was a huge variety in the research sites listed above, from organisms that cause illness in humans and animals to root nodules of a yam beans. About half of the hits that came up when BLASTing were for data that represented “uncultured” retrieval that used DNA-only information, none of these hits were listed above. This goes to support that many of the organisms living in the environment have yet to be successfully cultured.