Effect of Deuterium Depleted Water on Life REU: Alexandria Haddad Mentor: Anthony Salvagno Advisor: Steve Koch http://alexhaddadnm.wordpress.com/
Open Notebook Science “ Open Notebook Science is the • Wordpress.com practice of making the entire – Great place to get started creating an online notebook. Easy to use, shortprimary record of a research project learning curve, and lots of options. publicly available online as it is • Mindmeister.com recorded.” – Wonderful tool for project planning • Google Docs • Inception • DropBox.com • Planning • FigShare.com • Protocols – Post all your data sets online • Equipment • Slideshare.com • Data • Social Media • Conclusions – Facebook • Collaboration – Google + • Reproducible – Twitter
Understanding how water - particularly Hydrogen isotopes - affect biomolecular interactions and living cellsWe want to better understand how water with varyingamounts of D2O affects life forms.At what point does D2O become toxic to life?What is the mechanism of Deuterium/Hydrogen exchange?Since D2O occurs naturally, has life evolved to need it in some amount?
Deuterium - Hydrogen Isotope Common Hydrogen Water is the most abundant resource on the planetVs. Deuterium (Heavy Hydrogen) Naturally occurring water has about a 17mM (millimolar) concentration of deuterium Water (H2O) Vs. “Heavy” Water (D2O) Hydrogen has one proton and one electron, with an atomic mass 1 Deuterium has one proton, one neutron, and one electron with an atomic mass 2
Repeating CrumleyIn 1950 Helen A. Crumley et alperformed an experimenttesting plant seed growth invarying amounts of deuteriumoxide (D2O). They used H2Oand 33%, 66%, and 99% D2Omixtures with H2O. Theydiscovered that growth rateswere drastically slower in Influence of ordinary water, 33%, 66%, and 99% deuterium oxide on tobacco seed germination. Countsincreasing amounts of D2O. made at daily intervals for 39 days. (From Crumley, Fig 3)[Original Paper:http://iweb.tntech.edu/sstedman/JTAS%2025-3.pdf ]
Repeating Crumley EXPERIMENT DETAILS Our research was a repeat of the Crumley, In later experiments we added arabidopsis et al with the following changes: (mustard) seeds. They also used a variety of plant species: They placed the seeds on wet cloths, we• Tobacco submerged our seeds in sealed analyslides• Clover to better control the exchange of• Radish• Kentucky bluegrass deuterium. We used two species of Tobacco seeds: They used 100 seeds per experiment, we• Havana used around 30 – 40 seeds (the seeds are• Virginia Gold #1 really small and hard to count).
Repeating Crumley EXPERIMENT DETAILS (CONT) They reported their results in terms of percentage of germination, but the paper wasn’t clear how they calculated the percentages. Our results are a percentage of the seeds that germinate. The pink box shows a germinating seed. typical examples, of no germination, beginning The orange box highlights germination, obvious germination, etc what a non-germinating seed.All research information… success, failures, and procedures can be found online at: http://alexhaddadnm.wordpress.com/category/rc-repeated-cromley-experiment/ http://research.iheartanthony.com/tag/d2o-effects-on-life-2/ http://research.iheartanthony.com/category/water-type-effects-on-organism-growth/rc2/?orderby=date&order=ASC
D2O – Repeating Crumley (RC) 6 two-week experiments, the RCD experiment took 35 daysRC1 Five water samples: RC5 No Arabidopsis, eight • DI, DDW water samples as previous • 33%, 66%, and 99% D2O RCD Six analslides of D2ORC2 Eight water samples: • DI control w/out seeds with tobacco seeds • DI, DDW RCW Two species of tobacco • 33% and 66% D2O in DI • 33% and 66% D2O in DDW in four purified water types • 99% D2O (pure D2O) (eight samples): • CHTMRC3 Same setup as RC2 • RoDI purifiedRC4 Added Arabidopsis • Sigma Molecular Biology pure • Tissue Culture pure
FTIR Spectroscopy• Visually (to the naked human eye), all water looks the same• Spectroscopically water can be very different: – Can we notice a difference between the amounts of D2O in H2O (or vice versa) – Does DDW absorb D2O naturally over time? – Isn’t all deionized water the same? – What about D2O… does it change over time?• Special thanks to Dr. Sanjay Krishna and Stephen Myers who granted me access to and use of their laboratory’s FTIR, and graciously trained me on it’s use. Stephen was also especially helpful for spectroscopic interpretation.
Gilbert Lewis• Was the first to purify heavy water• Was the first to report that tobacco seeds do not grow in pure D2O• He hypothesized that life may have evolved a need deuterium – No one has explored this question yet!
Seeds Grown in DDWVirginia Gold seeds in DDW Dark Virginia seeds in tap water
E.Coli and Yeast Experiments1. Expectations: a. That neither organism would grow in 99% D2O b. DDW and DI growth would be the same2. Process: a. Make starter cultures: a. Make YPD and LB Broth b. Starter yeast and e.coli colonies c. Agar Plates and Broth
Growing E.Coli [Yeast] (excerpt from my online notebook)1. put on gloves – very important not to 5. Dispose of pipette tube in bio-hazard contaminate myself or the medium bin.2. get supplies: 6. Remove parafilm from agar plate. • 10mL tube and pipette 7. Using inoculating loop, get a single colony of e.coli [yeast] on loop. • inoculating loop, Green 10 x 1µL 8. Put loop in test tube and swirl for a • autoclaved test tube few seconds.• LB broth (pre-made… this is a separate 9. Dispose of loop in bio-hazard bin. process) [YPD broth] 10. Recover test tube.• agar plate with e.coli – LB Day 2 batch 11. Place test tube in incubator at 37° C [agar plate with yeast] [24° C].3. Remove cover from LB [YPD] broth 12. Re-cover agar plate and seal with and pipette 10mL of broth into test new parafilm. tube. 13. Place agar plate and LB [YPD} broth back in refrigerator.4. Re-cover test tube and broth.
Initial Setup – Nanodrop Readings We first wanted to get a spectroscopic reading of yeast and e coli grown in common water.
E. Coli Growth Over 4 Hours at 600 nm “continuous” data extrapolated from the 3 separate growth data setsgrowth rates of 3 dilutions of e. coli in LB broth (DI water)
What I’m taking with me• Research opportunities available – Awarded another NSF internship for the summer• Open Notebook Science and Open Access – This will be useful in all my future endeavors… even if I work somewhere that is classified, I now have a solid skill set and appreciation for keeping an electronic notebook.• Development of existing interpersonal skills• Appreciation and fascination for science that isn’t in my field of study• Relationships with some wonderful people …
AcknowledgementsThank you to everyone for providing me with the opportunity to experiencethe research field. This has been a wonderful experience for me.• NSF• CHTM• Dr. Marek Osinski• Linda Bugge• Dr. Steve Koch• Anthony SalvagnoImages provided by:• http://www.etftrends.com/2010/07/water-etfs-play-dwindling-natural-resource/• http://www.isowater.com/heavy-water/• Linda Bugge• Anthony Salvagno