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| Quantifying Stresses Endured by E. coli | Amanda D. Matherly1, Tiffany R. McElravy2, Melissa L. Deitrich2, Ashley L. Brady2, Anne L. Barrow2, Jessica Lake3, C. W. Vermeulen4 (1) Galileo Science Magnet High School, Danville, VA; (2) Jamestown High School, Williamsburg, VA; (3) Long Beach Senior High School, Long Beach, LI, NY; (4) Science-Projects.Com, Williamsburg, VA (ecoligist@yahoo.com) |
| As presented to the Amer. Soc. for Microbiology 23 May 2006 Atlanta, GA |
How does one quantify the health (or the sickness) of a culture so as to be able to compare different cultures? Use of the log-phase portion of the classic turbidimetrically derived growth curve has been called into question when comparing various E. coli cultures because it has been shown that oxygen deprivation stress begins at a threshold of about 2x107 cfu/ml, which is near the bottom of spectrophotometric sensitivity. Reported to the ASM this year were two quantitative methods for the measurement of stress. (1) The more stress endured by the cells, the higher the expression levels of their lac-operons, and (2) the more stress endured, the lighter (in specific gravity) the cells become. It was shown previously that specific gravity of whole E. cells was due to ribosome content, which varies over a three fold range. Both methods are indirect measurement of the cells' ATP pools.
↑ (All info about this poster is found on this webpage and links.)
Materials & Methods: Wild-type E. coli were grown in lactose-broths having various concentrations of NaCl, KCl and sucrose that would act at least as osmotic stressors. The expression levels of the lac-operon used the classic ONPG kinetics test, while the specific gravities of the cells were assayed using NaBr density-gradient equilibrium centrifugation.
Results: A direct linear relationship exists between lac-operon expression and the concentration of stressor. The expression of this gene is dependent on the reciprocal of [ATP] which is drained for use by the osmoregulation mechanisms of the cell. An inverse linear relationship between cellular specific gravity. Overall, the authors preferred the specific gravity method for several reasons - inexpensive, far faster, more precision, and being a equilibrium method, attention to the clock is not necessary.
Discussion: Both industry and chemotherapeutic manufacturers can use these methods to optimize their production. Researchers can, of course, make much more reliable comparisons be cultures.
"Science-Projects.com" encourages students, teachers and retirees to work collaboratively on original projects with an aim toward making presentations beyond science fair. This is the seventh presentation at the ASM made under this logo. The website is owned and operated by Dr. Vermeulen, emeritus microbiologist from the College of William and Mary in Williamsburg, Virginia.