Geochemistry & Ecology of Red Mat Systems (GERMS)

Undergraduate Summer Research Program

Week Five

DNA Sequence Analysis

Sequencing Reaction Set – Up: Site, Hillside

Step 1: Fill tubes with appropriate TGAorC stop nucleotides
Step 2: Mix the cocktail with the template samples of our clones. Cocktail contains dNTPs and Taq polymerase.
Step 3: Mix the newly formed cocktail of dNTPs, Taq, and clone DNA template to the tubes containing the stop nucleotides. Finally place in PCR.

Preparing Gels:

Step 1: Washing the plates and then drying with 95% ethanol. Must be really clean.
Step 2: Elizabeth is adding ddH2O to 0.05g APS (catalyst that will polymerize the gel) to bring the volume to .5mL.
Step 3: Preparing the reagents for the gel. Weighing out 17.0g of Urea to be mixed with ddH2O until the Urea is just saturated.
Step 4: Heating the Urea and ddH2O on a hot plate to liquefy. A stir bar is added to help with even melting.
Step 5: While the gel is heating we prepared the plates for pouring. The first step is to lay the spacers so that they are flush to the sides of the glass.
Step 6: Once the spacers are in place the clamps can be added to make sure that no gel leaks out. The clamps need to be tightened just barely.
Step 7: The prepared plates are now placed at an angle so that when the gel is poured it will flow down. The paper at the bottom is there to catch any gel that runs off. In order to stop the flow of gel the plates will be lowered to a flat position on the table.
Step 8: Coming from the heating plate, the liquefied gel needs to be brought up to a volume of 40mL, therefore it’s being poured into a graduated cylinder.
Step 9: Elizabeth and I are bringing the volume of the gel up to 40mL.
Step 10: Jana and I are adding the prepared catalysts, APS and TEMED to the gel. In preparation for pouring.
Step 11: The green tip is where the gel is being poured from and I am tapping the plates to make sure that no air bubbles get caught in between the plates. The oxygen would ruin the polymerization process.
Step 12: After the gel has been poured and is in the flat position the comb is placed in upside down.
Step 13: With the comb in, the final clamp is put on at the top and the gel is left alone for an hour and a half to polymerize and cure.
Step 14: After the gel has polymerized it can be stood up and the excess gel in between the comb can be removed with ddH2O.
Step 15: The comb has been removed and the back plate has been cleaned and the excess gel removed. Now any excess gel in the well needs to be removed with TBA buffer.
Step 16: The comb is placed in teeth down and should poke into the gel no more than 1mm.
Step 17: Liz and I wash the outside of the plates with 95% ethanol before loading into the DNA sequence machine. The outside must be clean for the machine to read the gel in between the glass.
Step 18: Liz pours the buffer that we made into the lower buffer chamber. It will also be poured into the upper buffer chamber.
Step 19: Liz places the cap with the electrode on the lower buffer chamber.
Step 20: The top buffer chamber is being capped and the electrodes connected by Liz. Next we will close up the machine and wait for 15 minutes while the DNA sequencer does a pre-run. While that is going we will gather our sequencing tubes for denaturing.

Reaction Finish and Load:

Step 1: Remove sequencing DNA from freezer.
Step 2: Loading DNA samples into PCR machine for a denaturing time of 3 minutes.
Step 3: Finally, load the denatured DNA into the gel in the DNA sequencing machine.

Reading, Editing and BLASTing Sequences

SEQUENCE INFORMATION

Clone Number Analyzed: HR1

Edited Sequence:

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.

Top Accession #	Organism Name	Source
AB069654	Bradyrhizobium	yam bean
AB069652	Rhizobium	yam bean
AY077753	Obesumbacterium proteus	unknown
AJ431219	Proteobacterium BHI60-11	Novel bacterial lineages from enrichment cultures with Alvinella pompejana white tubes collected on East Pacific Rise at 13oN :effects of temperatures and media
M22510 M22467	C.violaceum	Chromobacterium, Eikenella, Kingella, Neisseria, Simonsiella and Vitreoscilla species comprise a major branch of the beta group Proteobacteria by 16S rRNA sequence comparison: transfer of Eikenella and Simonsiella to the family Neisseriacae (emend.)

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?) From the blasting data above there are two types of bacteria that come from the same source, Bradyrhizobium and Rhizobium. The other three are all from varying sources, one of which is unknown. This was a fairly good sequence because all of the top five hits were near the top of the list, however there were a lot of sequences that the program recognized that do not relate to the bacteria normally found in the mats.

Clone Number Analyzed: HR2

Edited Sequence:

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.

Top Accession #	Organism Name	Source
AJ441242	Uncultured CFB	mucous secretions of the hydrothermal vent polychaete Paralvinella palmiformis
AJ318130	Uncultured bacterium	High bacterial diversity of a waste gas-degrading community in an industrial biofilter
L24106	Mycoplasma meleagridis	turkey pathogens
AF221114	Mycoplasma iners isolate PG30(T)	Mycoplasma lipophilum
AF221113	Mycoplasma columbinum isolate MMP-1(T)	Mycoplasma lipophilum

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?) This blast data was all over the board in terms of sources, there are only two that are the same. This was also fairly good sequencing because I had many hits that contained the 16S RNA at the top, it was only toward the middle and end that there was more and more sequences that didn’t match anything I was looking for; such as the sequences for HIV, tomatoes, mouse muscles, etc.

Clone Number Analyzed: HR3

Edited Sequence:

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.

Top Accession #	Organism Name	Source
AF121886	Bacterium 2BP-6	Community analyses of sulfidogenic 2-bromophenol-dehalogenating and phenol-degrading microbial consortia
AJ006853	sulfate-reducing bacterium mXyS1	Unknown
AJ306764	Uncultured bacterium	Unknown
AY077753	Obesumbacterium proteus	Unknown
AF002671	Desulfobacca acetoxidans	sulfidogenic granular sludge

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?) In this sequence, I had good data in terms of getting 16S RNA bacteria, however, most of the sources were unknown. Also, the Obesumbacterium proteus was found in another of my earlier cultures, HR1. This is a good representation of consistency within the cultures.

Clone Number Analyzed: HR4

Edited Sequence:

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.

Top Accession #

Organism Name

Source

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?) Culture either sequenced the vector only or was a bad insert, never-the-less I got no data on any 16S bacteria.

Clone Number Analyzed: HR9

Edited Sequence:

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.

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 : effects of temperatures and media
AB064705	Uncultured bacterium	The microbial community in an uncoupler 2,4-dinitrophenol-digesting reactor as reactor as revealed by molecular analysis
AB069653	Bradyrhizobium	yam bean
AJ431341	Uncultured firmicute	Microbial diversity in ikaite tufa columns: an alkaline, cold ecological niche in Greenland
AJ296574	Uncultured bacterium GR/GS-296.II.265	Bacterial diversity in drain waters of several uranium waste piles

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?) I didn’t have a lot of high numbers on this culture, but I was able to find 5 sequences that had the 16S RNA, however, because I didn’t have very high numbers I ended up finding them all throughout the list. There is some consistency with the first culture in that the same organism, Bradyrhizobium, was found. This was a fairly good sequence.