Team:UC Davis/Design

Can we detect enzyme inhibition in waste water?

In order for our show we had a functional prototype, we needed to show enzyme inhibition in waste water. We performed this experiment using triplicates of 15 nM P. falciparum FabI. It appears as if life is a bit slower in waste water…

This shows that our biosensor works in waste water!

  • Confirm inhibition data in waste water correlates to known levels of triclosan in a wide variety of waste water samples
  • Continue rounds of enzyme engineering to enhance another 60-fold (~10 more 1.5 folds… or 1 60-fold)
  • Forward predictions based on unknown samples where the biosensor, ELISA, and MS are used in parallel

When we first started our project, we took a trip to our local Safeway to catalog products containing triclosan. We discovered that many products had already phased out triclosan; some labels even read “Triclosan Free.” Although triclosan had been removed from products, many of them had simply replaced it with a different antimicrobial.

This trend reminded us of what Arlene Blum told us about how when chemicals are removed from use manufacturers look for a replacement; but because these chemicals need to serve similar functions they often have similar structures, and thus similar consequences. What results is a cycle whereby one toxic chemical is replaced by another toxic chemical.

We didn’t want to raise fear over triclosan use and contribute to this cycle. Instead we wanted to raise awareness around appropriate chemical use and reduce the use of chemicals in cases where there is no proven benefit.

This lead us to supplementing our triclosan biosensor with an, “antimicrobial footprint app,” to get consumers thinking about whether antimicrobial agents are even warranted in consumer products.

We designed our app as a heuristic to raise awareness about the unnecessary ubiquity of antimicrobials in consumer products. In the app, the user can click on an “About” tab to learn more about antimicrobials and how to be a responsible consumer. They can then go on to calculate their “Antimicrobial Footprint.” The user is able to click on antimicrobial containing products that they use, and see how it affects their total footprint. After using the app’s antimicrobial calculator to calculate their footprint, the user can submit their footprint along with their location. On the final page of the app the user is able to see how their footprint compares to the average footprint of other users. The submitted data is used to calculate this average, as well as to create a heat map of antimicrobial usage in the United States. This is another deliverable that users can look at to become more educated consumers.

space space space space

How It Works:
To create the antimicrobial calculator we found data on the levels of triclosan in selected consumer products, given in g triclosan/g products. We also found data on the daily use rates of consumer products, given in g triclosan/day. By combining this information we were able to calculate the users’ “antimicrobial footprint,” in grams triclosan/day. The app will also give you this metric in grams triclosan/year.

space space space

Lesson Plan Sources:
Ref #.1 J. W. Pellegrino, M. R. Wilson, J. A. Koenig, A. S. Beatty, Developing assessments for the next generation science standards (National Academies Press, 2014).
Ref #.2 Russell, J. & Hollander, S. (1975). A biology attitude scale. The American Biology Teacher, 37 (5), 270-273. via (
Ref #.3 Alwin, DF & Krosnick JA. 1991. The reliability of survey attitude measurement the influence of question and respondent attributes Sociological Methods & Research 20, 139-81.

Biosensor Sources:
[1] J, Regös, Zak O, Solf R, Vischer WA, and Weirich EG. "Antimicrobial Spectrum of Triclosan, a Broad-spectrum Antimicrobial Agent for Topical Application. II. Comparison with Some Other Antimicrobial Agents." National Center for Biotechnology Information. U.S. National Library of Medicine, 1979.
[2] Kini, Suvarna, Anilchandra R. Bhat, Byron Bryant, John S. Williamson, and Franck E. Dayan. "Synthesis, Antitubercular Activity and Docking Study of Novel Cyclic Azole Substituted Diphenyl Ether Derivatives." EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY. N.p., May 2008.
[3] McMurry, Laura M., Margret Oethinger, and Stuart B. Levy. "Triclosan Targets Lipid Synthesis." Nature 394 (1998): 531-32.
[4] Heath, R. J. , Yu, Y.-T. , Shapiro, M. A. , Olson, E. & Rock, C. O. J. Biol. Chem. 273, 30316–30320 (1998)
[5] RP, Massengo-Tiassé, and Cronan JE. "Diversity in Enoyl-acyl Carrier Protein Reductases." Cell Mol Life Sci. (May 2009)
[6] RJ, Heath, Rubin JR, Holland DR, Zhang E, Snow ME, and Rock CO. "Mechanism of Triclosan Inhibition of Bacterial Fatty Acid Synthesis." J Biol Chem (April 1999)
[7] Ward, Walter. "Kinetic and Structural Characteristics of the Inhibition of Enoyl (acyl Carrier Protein) Reductase by Triclosan." Biochemistry (1999 Sep 21)
[8] Kapoor, Mili. "Slow-tight-binding Inhibition of Enoyl-acyl Carrier Protein Reductase from Plasmodium Falciparum by Triclosan." Biochem (2004 August 1)
[9] Surolia, Namita, and Avadhesha Surolia. "Triclosan Offers Protection against Blood Stages of Malaria by Inhibiting Enoyl-ACP Reductase of Plasmodium Falciparum." Nature Medicine (2001)
[10] Marcinkeviciene, al, (2001). "Enoyl-ACP Reductase (FabI) of Haemophilus influenzae: Steady-State Kinetic Mechanism and Inhibition by Triclosan and Hexachlorophene." Archives of Biochemistry and Biophysics 390(1): 101-108.
[11] Courtney Slater-Radosti, Glenn Van Aller, Rebecca Greenwood, Richard Nicholas, Paul M. Keller, Walter E. DeWolf, Jr, Frank Fan, David J. Payne, and Deborah D. Jaworski Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus J. Antimicrob. Chemother. (2001) 48 (1): 1-6. doi: 10.1093/jac/48.1.1
[12] Mechanism and Inhibition of saFabI, the Enoyl Reductase from Staphylococcus aureus Hua Xu, Todd J. Sullivan, Jun-ichiro Sekiguchi, Teruo Kirikae, Iwao Ojima, Christopher F. Stratton, Weimin Mao, Fernando L. Rock, M. R. K. Alley, Francis Johnson, Stephen G. Walker and Peter J. Tonge Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, School of Dental Medicine, Stony Brook University, Stony Brook, New York 11794, Department of Infectious Diseases, International Medical Center of Japan, Tokyo 162-8655, Japan, and Discovery Biology, Anacor Pharmaceuticals Inc., Palo Alto, California 94303
[13] Hoang TT, Schweizer HP. 1999. Characterization of Pseudomonas aeruginosaenoyl-acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis. J. Bacteriol.181:5489–5497.
[14] Parikh, S. L., Xiao, G. and Tonge, P. J. (2000) ‘Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid’,Biochemistry, Vol. 39, No. 26, pp.7645-7650.
[15] Massengo-Tiassé, R. P., and J. E. Cronan. 2009. Diversity in enoyl-acyl carrier protein reductases. Cell. Mol. Life Sci.66:1507–1517.
[16] Dayan FE, Ferreira D, Wang YH, Khan IA, McInroy JA, Pan Z
(2008) A pathogenic fungi diphenyl ether phytotoxin targets plant enoyl (acyl carrier protein) reductase. Plant Physiol 147: 1062–1071
[17] Liu N, Cummings JE, England K, Slayden RA, Tonge PJ. 2011. Mechanism and inhibition of the FabI enoyl-ACP reductase from Burkholderia pseudomallei. J. Antimicrob. Chemother. 66:564–573. 10.1093/jac/dkq509
[18] Chalew T. E., Halden R. U. (2009). Environmental exposure of aquatic and terrestrial biota to triclosan and triclocarban. J. Am. Water Works Assoc. 45, 4–13. 10.1111/j.1752-1688.2008.00284.x
[19] Eriksson M, Johansson H, Fihlman V, Grehn A, Sanli K, Andersson MX, Blanck H, Arrhenius Å, Sircar T, Backhaus T. (2014) Long-term effects of the antibacterial agent triclosan on marine periphyton communities. PeerJ PrePrints 2:e489v1 [20] "Guidelines for High School Physics Programs." HS Guidelines.
[21] Corp., Sigma-Aldrich. Acetyl Coenzyme A Trilithium Salt (A2181) - Product Information Sheet (n.d.): n. pag. Sigma.
[22] Vick JE, Clomburg JM, Blankschien MD, Chou A, Kim S, Gonzalez R.Escherichia coli enoyl-acyl carrier protein reductase (FabI) supports efficient operation of a functional reversal of β-oxidation cycle. Vol. 269, No. 8,Issue of February 25, pp. 5493-5496, 1994 The Journal of Biological Chemistry, 269, 5493-5496.
[23]. Pidugu, L. S., M. Kapoor, N. Surolia, A. Surolia and K. Suguna (2004). "Structural basis for the variation in triclosan affinity to enoyl reductases." J Mol Biol 343(1): 147-155.
[24] Links to purchase NADPH and NADH:,
[25] White, S. W., J. Zheng, Y. M. Zhang and Rock (2005). "The structural biology of type II fatty acid biosynthesis." Annu Rev Biochem 74: 791-831.
[26] Rafferty, J. B., J. W. Simon, C. Baldock, P. J. Artymiuk, P. J. Baker, A. R. Stuitje, A. R. Slabas and D. W. Rice (1995). "Common themes in redox chemistry emerge from the X-ray structure of oilseed rape (Brassica napus) enoyl acyl carrier protein reductase." Structure 3(9): 927-938.
[27] Elovson, J. and P. R. Vagelos (1968). "Acyl Carrier Protein: X. ACYL CARRIER PROTEIN SYNTHETASE." Journal of Biological Chemistry 243(13): 3603-3611.
[28] Perozzo, R., M. Kuo, A. Sidhu, J. T. Valiyaveettil, R. Bittman, W. R. Jacobs, Jr., D. A. Fidock and J. C. Sacchettini (2002). "Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase." J Biol Chem 277(15): 13106-13114.
[29] Johansson, C. H., L. Janmar and T. Backhaus (2014). "Triclosan causes toxic effects to algae in marine biofilms, but does not inhibit the metabolic activity of marine biofilm bacteria." Mar Pollut Bull 84(1-2): 208-212.
[30] Savile, C. K., J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, P. N. Devine, G. W. Huisman, and G. J. Hughes. "Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture." Science 329.5989 (2010): 305-09.
[31] J. B. Siegel et al., Science 329, 309 (2010)
[32] Bornscheuer, U. T., G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore and K. Robins (2012). "Engineering the third wave of biocatalysis." Nature 485(7397): 185-194.

Footprint App Sources:
Rodricks, Joseph V. "Triclosan: A Critical Review of the Experimental Data and Development of Margins of Safety for Consumer Products." Critical Reviews in Toxicology, 2010. Web.