This week I was ambitious and decided to drill four of my samples for Uranium dating. I only got two done, with lots of support from Eileen Embid. (The drill is pictured above)




There are studies that have already assessed the timing of travertine accumulation in the Rio Salado, the Grand Canyon, and Springerville, AZ (Sower et al., 2008; Embid et al., in prep.; Crossey et al., 2009). My study focuses specifically on the active spring deposits in the Tierra Amarilla anticline, just south of the Rio Salado and their correlation with results from the surrounding area.

To better understand the paleohydrologic processes and to estimate the growth record of the mound system, geologic mapping and U-series geochronology is being incorporated. The active and extinct mound springs we propose to study are morphologically similar to mounds elsewhere in the southwestern U.S., including in Springerville, AZ and the Grand Canyon.

U-series chronology of travertine samples will be determined in the Radiogenic Isotope Laboratory at the University of New Mexico. The chemistry lab and mass spectrometry lab are class 100 clean labs. The travertine samples will be cut and drilled samples will be obtained along growth bands with an automated drill. The carbonate powder, typically around 200 mg, will be dissolved in HNO3 and spiked with a mixed 229Th-233U-236U spike. You can see my fresh travertine powder above and Eileen is putting in a vial for safe keeping until January when we will run the samples in the lab.


The Micromass Sector 54 thermal ionization mass spectrometer with a high-abundance sensitivity filter will be utilized for all U and Th measurements (Rasmussen, 2001). The timing of travertine accumulation will be determined and than correlated with nearby accumulations (Springerville, AZ and Grand Canyon). We will define the time interval over which these springs have been active and these dates will relate to the surrounding region and the incision rates of the Rio Salado. We hypothesize that this region will provide unique dates that do not correlate with the Grand Canyon or the Springerville area (i.e., controls are local).

Eileen Embid just finished her Master's at UNM working with travertine from Springerville,AZ. If you want to see more information on her and the other graduate students in our research group here is our link: http://epswww.unm.edu/facstaff/lcrossey/PersonalPage/students.html

"Studies of simpler and more stable systems make it easier to observe patterns that may influence the way we think about more complex systems."

If you were ever curious about how certain bacteria survived the Cretaceous-Paleogene global extinction (extinction of the dinosaurs) read the following:
Astrobiology, vol. 9, Number 6, 2009, Harriet Jones, et al., Experiments on Mixotrophic Protists and Catastrophic Darkness.
In this paper several experiments were done to test the effect that 6 months of darkness and exposure to controlled amounts of DOC would do to mixotrophic bacteria. Turns out, they eat each other's dead bodies in order to survive. I am trying to imagine what a human would do if it was locked up with controlled nutrients with 1000's of other humans in the dark for 6 months... and I'm thinking "Zombies"? I guess we are not too far from the root of the tree, huh?

YSI Sonde

Two weeks ago I deployed two YSI sondes into the travertine mound spring located at the northern most point of the Tierra Amarilla anitcline. I left the instruments in the springs for three days. They recorded measurements of DO, pH, temperature, and conductivity every 2 minutes; one at 1m depth and the other at 5m depth. The goal for our research is to synchronously measure all parameters in these springs to hopefully predict what and how these springs are altered by climate and/or seismic events. In order to properly assess the data that I have from the Sonde deployments I need to incorporate diel cycling, hydrologic cycling, and maybe lunar cycling. I have a link to the GOSA wesite. The paper I'm currently reading refers to temperature measurements that were taken from Upper Geyser Basin in Yellowstone National Park with an instrument similar to ours. It was found that GEIs are lengthened when the water discharge is low. They also conclude that the GEIs are effected not only by the water that is provided by the warm subsurface, but also by the colder water that is provided by the geyser surface. The GOSA link is also an interesting link for anyone who wants to know more about how geysers work:
http://www.geyserstudy.org/
Also, here is a link to the USGS real-time water data for Firehole River: http://waterdata.usgs.gov/wy/nwis/uv/%20?site_no%20=%2006037500&agency_cd%20=%20USGS measurements include temperature fluctuations and discharge rates.
I might be able to use these data for an interpretation for my 1m vs. 5m depth measurements. So far it look as though the 1m depth water is retaining surface and subsurface water (recycling the water before discharging it to the outside of the mound), whereas the 5m depth water is constantly refueling from the subsurface. Or at least this is what I hypothesize.

Diatoms!

I made a tree for a Biology class this past semester from Dr. Andrea Porras-Alfaro http://sites.google.com/site/andreaporrasalfaro/and found out that a chloroplast gene AJ536457_1 Odentella sinensis (a cyanobacterium) was cloned and sequenced from a water filter that was collected from the mound spring at Tierra Amarilla in 2007.

I talked with a fellow graduate student this morning and we had a good conversation about diatoms! Diatoms are my second hobby, with microbes being my first. He wants to do some interdisciplinary work with my advisers and myself to investigate the role that diatoms have in the production of calcite. He looks at the isotopic configuration of the silica shell and determines where the oxygen in their bodies comes from. I am interested in the symbiotic relationship between microbes and diatoms and the possibility of HGT between the two. Our conversation prompted me to look up my old research paper for this class I took. I spent an hour or so today looking at the NCBI citations and turns out I forgot what it meant if this gene I sequenced was a chloroplast gene from Odentella sinensis. Odentalla sinensis is a chromista. So, a chromista is: http://www.ucmp.berkeley.edu/chromista/chromista.html. Turns out I needed a refresher on plastids as well. A plastid is: http://en.wikipedia.org/wiki/Plastid... if you are into wiki definitions. So, according to the NCBI results, the O. sinensis diatom species is living at the water's surface and we extracted, cloned, and sequenced its chloroplast gene that previous research has concluded is evidence for some sort of symbiont relationship -or- HGT.

There is quite a bit of work that has already been done on the horizontal gene transfer between certain mitochondria and plastids. One such study shows HGT between an algae and a diatom species (An exceptional horizontal gene transfer in plastids: gene replacement by a distant bacterial paralog and evidence that haptophyte and crypotphyte plastids are similar: BMC Biology: 2006: Rice and Palmer) Does this mean that there could be a gene in a diatom (the Odentalla sinesis) at Tierra Amarilla which was acquired through HGT from a photosynthetic bacterium?

Another interesting note: we cultured, yes, CULTURED diatoms and cocci bacteria on the same plate last year when we sampled at Soda Dam (above picture). How the heck does this happen? Well, it is Friday, so first thing Monday (after I PCR and ligate my own water filters for their long trip to WashU) I am going to pay a visit to Becky Bixby, UNM's own diatom specialist . As if I have time to pursue this, but I've been advised to keep at least one scientific hobby for my own sanity.

Woods Hole Oceanographic Institute Marine Microbial Biogeochemistry

I have got some more reading to do:
http://www.whoi.edu/science/MCG/people/kcasciotti/cv.htm
Main question I'm asking myself: How can I incorporate isotope geochemistry into my project? Secondary question: How do I get myself a job at Woods Hole?
http://www.whoi.edu/science/MCG/people/kcasciotti/research.html

But first- does anyone out there know how to do Unix? ARB is ridiculous!
http://www.arb-home.de/downloads.html
I am stuck on step #7:
http://greengenes.lbl.gov/mlds/

For now, I'll just reminisce about the ocean and re-read the ARB instruction manual.

MOBIO experienced User Protocol

Extraction of the water filters that came from the surface waters of the Eastern Twin Mound (Above picture) went well. PCR of the DNA will take place tomorrow. The protocol for yesterday's extraction changed. I used the soil extraction kit instead of the phenol:cholorform protocol from Dr. Vesbach's lab.

Protocol:
DNA Extraction from water sample (using Mo Bio Power Soil Kit catalog #12888)

Sample should have sucrose lysis buffer added in the field and be stored at -80 degrees Celsius. Be sure to do a negative control to ensue sterile technique. Everything added to the sample should also be added to the negative control with exception of the actual sample. All centrifuge spin times are done at 10,000rcf at room temperature.

1. Place sample on ice and allow to thaw completely.
2. Add 60 uL of solution C1 to the Mo Bio Powerbead tubes. Add sample to the tube. Be sure to add as much as possible. Leave room in the top of the tube.
(C1 contains SDS and other disruption agents required for complete cell lysis. In addition to aiding in cell lysis, SDS is an anionic detergent that breaks down fatty acids and lipids associated with the cell membrane of several organisms. It if gets cold, it will form a white precipitate in the bottle. Heating to 60 degrees Celsius will dissolve the SDS and will not harm the SDS or the other disruption agents.)
3. Vortex for 1 minute.
4. Centrifuge for 30 seconds.
5. Pour supternatant to a sterile 2ml microcentrifuge tube.
6. Add 250 ul of solution C2, vortex and incubate at 4 degrees Celsius for 5 minutes.
(Solution C2 contains a reagent to precipitate non-DNA organic and inorganic material including humic substances, cell debris, and proteins; these material can contaminate the DNA purity and inhibit further analysis of the gene in question)
7. Centrifuge for 1 minute.
8. Without touching the pellet transfer 600 ul (or less) of solution to a new sterile 2 ml microtube.
9. Add 200 ul of solution C3, vortex and incubate at 4 degrees Celsius for 5 minutes.
(Solution C3 is a second reagent that removes contaminating organic and inorganic material)
10. Centrifuge for 1 minute.
11. Transfer 750 ul (or less) of solution to a new sterile 2 ml microtube. (Do not touch the pellet)
12. Add 1200 ul of solution C4 and vortex.
(Solution C4 is a saline solution that allows the DNA to attach to the silica inside the Spin Filter.)
13. Transfer 675 ul of the solution to a spin filter and cetrifuge for 1 minture. Decant the flow through (Remove the liquid in the bottom of the spin filter).
14. Repeat step 13 until no solution is left.
15. Add 500 ul of solution C5 and cetrifuge for 30 seconds. Discard the flow through.
(Solution C5 is an ethanol based wash solution. The ethanol cleans the DNA that is attached to the silica filter of any excess salt, humic acid, and other contaminants.)
16. Centrifuge an additional 30 seconds to remove any excess C5 from the silica filter.
17. Transfer the filter to a labeled sterile 2 ml microtube.
18. Add 30 ul of solution C6 to the membrane filter. Be sure to get the solution onto the actual white membrane in teh filter.
(Solution C6 is a sterile elution buffer (10 mM Tris) that wets the membrane and effectively releases the DNA from the membrane.)
19. Centrifuge for 30 seconds.
20. Discard teh filter and store the tube containing DNA at -20 degrees Celsius.

Currently reading:
http://schloss.micro.umass.edu/mothur/The_effects_of_eutrophication_on_the_structure_of_bacterial_biofilms

Stranger in a Strange Land, Robert A. Heinlein

Back to the Lab

Microbiology Update:
Tomorrow is my first day back in the molecular laboratory. Ian Mcmillan will be showing me how to extract DNA from the filters that I collected cells in two weeks ago. The filters have been sitting in the -80 Celsius freezer since their collection. Below is the protocol written by Justine Hall and Kendra Mitchell. I will be changing it with advisement from Dr. Northup by applying a bead beating method instead of gently 'inverting' the filter after the CTAB and pK are added.
My experiment for this month is to try the bead beating, the gentle inverting technique, and using a MoBio kit on the filters. I hope to determine which method will produce the best results from the waters I am working with.
Geochronology update:
I need to get my travertine samples from this past fall drilled and prepped for analysis the in clean lab. I need the U-series dates for my five samples to complete my cross section of the anticline.

Collecting community biomass from water

Sterivex GP 0.22um Filter unit – Millipore Cat. # SVGP01015.
60 ml syringe with luer lock
3 ml syringe with luer lock
SLB (20mM EDTA, 400mM NaCl, 0.7M sucrose, 50 mM Tris, pH 9.0)

These filters are designed for sterilizing solutions but are also large enough that we have collected biomass from “clear” water and successfully extracted DNA. The more cells that you collect, the better your chance of getting decent DNA. So, maximize the amount of water you filter. For Yellowstone boiling springs, we tried to filter at least a liter of water or until the filter clogged. Be very careful to keep your syringe and filter “sterile” while you are filtering the water. Don’t touch the end of the syringe or any part of the the filter. I used the packaging that both syringe and filter come in to keep everything sterile. You should be able to concentrate on not contaminating your sample because the procedure is very simple.

1. Cleanly open the syringe packaging
2. Suck up 60 ml of spring water. Caution: if your source pool is near or at boiling, the water is likely to boil in the syringe because you’ve lowered the pressure. This will cause very hot water to squirt out of the syringe. Make sure that you point the syringe away from your field mates and away from the pool.
3. Cleanly attach the filter to the syringe. I open the packaging just enough to get the syringe into the packaging and, holding the filter through the packaging, twist the syringe on.
4. Pull the filter and syringe out of the packaging.
5. Push the water through the filter
6. Cleanly remove the filter from the syringe. Again I never touch the filter directly, only through the packaging.
7. Go back to step 2 another 15 times or until the filter clogs
8. Once you have clogged the filter, push all the water out of the filter by sucking air into your syringe and blowing it through the filter.
9. Draw 0.5 ml SLB into a 3ml syringe
10. Very slowly push as much SLB into the filter as you can without it coming out the outflow. We are trying to lyse the cells that are stuck to the filter and preserve their DNA.
11. Leave the 3ml syringe on the inflow to seal that end of the filter
12. Jam a p10 pipette tip on the outflow of the filter. Melt the tip to seal it.
13. Place the whole unit-filter and syringe-in a whirl-pack. Store at –80 until you extract the DNA. We have held the filters at ambient temperature for up to 10 days before freezing.


DNA EXTRACTION FROM STERIVEX FILTERS
Extracting from Sterivex filters requires only slight modification of the mocat DNA extraction. I have always treated sterivex as if they were 200 μL samples, which makes it possible to fit all of the reagents in the filter while still getting a good DNA yield. If you have more than 200ul SLB in your filter, you could remove all but 200ul and store the rest at –80 as a backup. All reagent volumes in this protocol are written for a 200 μL sample, but you may have to adjust them if you have a lot of residual water in the filter. This procedure can be performed on aerodisk filters, but it is a lot more tedious and will likely result in low DNA yields due to spillage.

Solutions
1. CTAB Buffer: 1% CTAB, 0.75 M NaCl, 50 mM Tris pH 8, 10 mM EDTA
2. Proteinase K
3. 20% SDS
4. phenol:chloroform:isoamyl alcohol (25:24:1) or chloroform:isoamyl alcohol (24:1)
5. chloroform
6. 3 M NaOAc
7. absolute ethanol
8. 70% ethanol
9. 10 mM Tris, Filter sterilized

Procedure:
1. Attach a p10 pipet tip to the small end of the filter. Melt the open end of the tip to close it. This is not a perfect fit, so make sure the tip is really on and thoroughly melted closed before adding any reagents.
2. Remove the syringe and add 400 μL CTAB and proteinase K to a final concentration of 100 ug/ml. This must be done SLOWLY as the reagents get bottlenecked at the top of the filter. A bubble may form in the neck of the filter – you can try to pop it or work around it. Replace syringe (can be used to blow reagents into the filter after they are all added).
3. Incubate for 1 h at 60 C. Filters can be rubber-banded to a rotator or you must periodically invert them.
4. Add SDS to a final concentration of 2%. Again, add reagent slowly and watch for bubble formation. Replace syringe.
5. Incubate for 1 h at 60 C.
6. Take syringe off and push all of the air out of it. Replace syringe, turn Sterivex upside down, and suck all of the lysate into the syringe. You may need to remove the pipet tip once all lysate is out of it. Put all lysate into an epi-tube for the rest of the extraction.
7. Extract with an equal volume of phenol:chloroform:IAA (24:24:1) or chloroform:IAA (24:1).
8. Extract twice with an equal volume of chloroform if using phenol:chloroform:IAA.
9. Add 0.1 volumes of 3M NaOAc, invert gently several times, add 2 volumes of 95% ethanol (Note: it may be necessary to split your sample in half to accommodate volumes)
10. Precipitate for 1h to overnight at -20 C
11. Spin for 45 minutes at 14 K rpm
12. Wash with 70% ethanol, spin for 30 minutes
13. Speed-vac to dry pellet, resuspend in 50 ul of filter sterilized dd water or 10 mM Tris, pH 8.0

My first Ragnar Relay

I am running the 1st, 13th, and 25th leg of the Ragnar Relay in Utah this Friday and Saturday. The race starts in Logan and ends in Salt Lake City, Utah. There are a total of 11 team members and 186 miles to cover. Above are the logistics for each of my legs. I'm only doing 20 miles, and my legs are flat or downhill. Woo-hoo!

NSF Comments

I applied for the NSF Graduate Research Fellowship Program last October. My application was reviewed by a panel of academic experts in the science, mathematics, or engineering discipline that identified in my chosen field of study. These are the comments that were made about three essays written by myself, and three letters of recommendation by my advisor, Dr. Diana Northup, and Dr. Anna-Louise Reysenbach. I rank in the 70th percentile for the range of applicants in the year 2008. I will try again this year, and use these comments to strengthen my personal statement, my future research plans, and my previous research experience. The NSF JAM 2009 Conference also helped me wrap my mind around the specific things that the NSF panels are looking for with these types of applications. Better luck this year!
Overall Assessment of Broader Impacts: Very Good
Explanation to the applicant:

By participating in a variety of undergraduate research opportunities and later participating in various out-reach activities you seve as an effective "scientific ambassador" within your community. Your application could be strengthened by elaborating a little more completely on your professional goals for the future
Overall Assessment of Intellectual Merit: Very Good
Explanation to the applicant:

Your previous research record is tied logically to your current investigation. Your research plan to compare microbial communities in marine and terrestrial settings is both worthwhile and original. Your application can be strengthened by providing more information on why/how you would expect these communities to compare.
Overall Assessment of Intellectual Merit: Good
Explanation to the applicant:

You are certainly a motivated, talented student and one who has moved forward in spite of facing adverse challenges. It is noted coming from a rural reservation area and accomplishing what you have thus far - is commendable. It is clear from your previous research experiences you have the passion, motivation and interdisciplinary understanding to plan and conduct a research project. You have built on previous research experiences to develop an original research project. It is noted in your letter of reference your ability to work independently as well as on a team and your development as a student. There is value to your research findings for a number of disciplines. You have worked to build your capacity in a number of science areas - to fully undertake the research proposal. It seems you have access to necessary resources - and have taken advantage of the STEM enrichment opportunities at the University of New Mexico (LSAMP, McNair etc.) to ensure your intellectual capacity in your Ph.D. You have drive and passion and these are critical traits for a researcher - partnered with your cultural background - there exists opportunities to support the oral history/Indigenous knowledge of the Navajo Nation. Your proposal would be strengthened by a stronger academic record as well as assurance you have access to sufficient resources.

Overall Assessment of Broader Impacts: Very Good
Explanation to the applicant:

You have been an active participant in programming focused on increasing the number of under-represented students and professionals in STEM. You have followed the pathway and it is noted you are maintaining active involvement in the next generation of students moving forward. The research experiences in England and the Atlantic Ocean sound to have created a motivation to pursue a Ph.D. It is understood you have developed a more global context to the broader impact of your research. You have developed a strong dissemination plan and have worked to present your research at local and national levels. Your proposal would be strengthened and more competitive with additional letters of support. Your letters of reference should provide specific examples of your research work, aptitude for and skills acquired during the experience.

Second day at Tierra Amarilla

The second day of sampling went well. Two springs, Iron Spring and Blowhole were successfully sampled. Three more springs were supposed to be sampled from, but were dry. On a trip to the anticline in November, these springs were still active. The change in water level is very interesting. I will return in July and again in the fall to see if the water level rises or recedes.
I will bring a YSI monitor out to the field in July (hopefully) to record fluctuations in pH, T, DO, and conductivity.
There are two springs located along the core of the anticline that have low water levels. The water is about 5m down from the surface. We think that the water in these holes is mainly from runoff and rain. The CO2 is still bubbling in them, but the DO, pH, T, and Conductivity are drastically different from the other springs (which we know are sourced from the subsurface) and much more similar to rain water. There might be a gaseous leak that is not correlated with the aquifer?
Photos from top: View from the trail of the southern terminus of the anticline, Blowhole spring, Minas Tirith, Iron spring, Mini Minas Tirith, Pressure Ridge with the Nacimiento in the background. All photos are courtesy of Ara Kooser. He has more photos at his flicker site:
http://www.flickr.com/photos/akooser/
Currently reading:
Redox Processes and Water Quality of Selected Principal Aquifer Systems, McMahon and Chapelle, Groundwater vol. 46, no.2, 2008

San Ysidro

Sampled seven spring this week. Looked at pH, T, Conductivity, Total Dissolved Solids, Dissolved oxygen, and collected water samples for major anion and cation analyses, along with alkalinity, nutrient content, and inorganic and organic content. I also collected microbial cells in sterivex filters for later extraction at variable depths in two "key" springs.