1. Effect of Deuterium Depleted Water on Life REU: Alexandria Haddad Mentor: Anthony Salvagno Advisor: Steve Koch http://alexhaddadnm.wordpress.com/
2. 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
3. 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?
4. 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
5. 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 ]
6. 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).
7. Repeating Crumley EXPERIMENT DETAILS (CONT) They reported their results in terms of percentage of germination, but the paper wasnt 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
8. 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
9. Repeating CrumleyEXPERIMENT DETAILS (CONT)
10. Koch Lab RC Results
11. 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? Isnt 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 laboratorys FTIR, and graciously trained me on its use. Stephen was also especially helpful for spectroscopic interpretation.
12. FTIR DDW absorption of D2O
13. 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!
14. Seeds Grown in DDWVirginia Gold seeds in DDW Dark Virginia seeds in tap water
15. 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
16. 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 1L 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.
17. Initial Setup Nanodrop Readings We first wanted to get a spectroscopic reading of yeast and e coli grown in common water.
18. E. Coli Growth Over 4 Hours at 600 nm continuous data extrapolated from the 3 separate growth data setsgrowt