Tim Riedel

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Directed Evolution of a Halotolerant E. Coli : 

Directed Evolution of a Halotolerant E. Coli Timothy E. Riedel and Andrew D. Ellington Abstract: We are using the technique of directed evolution to create a halotolerant strain of Escherichia coli. We see this as a first step towards evolving an organism capable of surviving under Martian surface conditions. The initial E. coli strain is capable of strong growth in LB broth with up to 1.0M NaCl added. After many rounds of selection, we have isolated a population that prefers to grow in LB broth with at least 3.0M NaCl added. We plan to continue the serial dilution protocol and will augment it with mutagenesis techniques if necessary. Once we fully establish the directed evolution protocols on this model system, we plan to use them on candidate organisms to expand any preexisting desirable traits for survival on Mars. Institute for Cellular and Molecular Biology, The University of Texas at Austin Conclusions: Through serial dilution selection, we have isolated an E. coli population that prefers growth in at least 3 times the starting salt concentration. We have demonstrated that directed evolution is a useful technique for selecting traits desirable for microorganism survivability on Mars. Directed evolution may prove to be an important organismal engineering platform for applications such as terraforming, planetary protection, and microorganism detection systems. Future Work: We are continuing to develop and optimize the serial dilution selection protocol with the ultimate goal of evolving a strain capable of growth in a saturated NaCl solution. We plan to characterize the genomic changes in the evolved strain using microarray technology. We would like to expand the directed evolution effort to include selection for low temperature and high radiation tolerance. We plan to start these experiments on candidate organisms already exhibiting desirable Martian traits. References: Bacher, J.M.; Ellington, A.D. (2002) The directed evolution of organismal chemistry: unnatural amino acid incorporation. In Translation Mechanisms. Lapointe, J., Brakier-Gingras, L., eds. Landes Bioscience and Eurekah.com Boer and Schmidt-Dannert. Engineering microbial cells to produce new chemical compounds. Current Opinion in Chemical Biology. 2003, 7:273–278 Clark, B.C., Baird, A.K., Rose Jr., H.J., Keil, K., Castro, A.J., Kelliher, C.W., Rowe, C.D., and Evans, P.H. (1976). Inorganic Analyses of Martian Surface Samples at the Viking Landing Sites. Science. 194: 1283-1288. Holmberg, N., Lilius, G., Bulow, L. (1994). Artificial anitfreeze proteins can improve NaCl tolerance when expressed in E. coli. FEBS Letters, 349:354-358. Lobitz, B., Wood, B.L., Averner, M.M., and McKay, C.P. (2001). Use of spacecraft data to derive regions on Mars where liquid water would be stable. PNAS. 98(5): 2132-2137. Reider, R., Economou, T., Wanke H., Turkevich, A., Crisp, J., Bruckner, J., Dreibus, G., and McSween Jr., H.Y., (1997). The Chemical Composition of Martian Soil and Rocks Returned by the Mobile Alpha Proton X-ray Spectrometer: Preliminary Results from the X-ray Mode. Science. 278: 1771-1774. Directed Evolution: Directed evolution is a powerful technique for enhancing existing or developing novel traits in nature (Boer 2003). Typically used to optimize a particular protein property, directed evolution can also be employed on the whole organism scale (Bacher 2002) . At the heart of the technique is the application of selection pressure on a pool of organisms. Some portion of this pool will be able to cope with the selection pressure better (grow faster) than the rest and will begin dominate the population. For this project we are exploring this technique and it's relevance to developing an organism capable of survival on the planet Mars. We believe such an engineered Martian will need water, and because the most likely stable water on Mars contains high concentrations of salt (see "life on mars" at right), we have begun this project by selecting for a salt tolerant E. coli. Materials and Methods: The directed evolution process has been carried out on an initial E. coli INVαF' strain transformed with the pET28c(+) vector (Novagen) carrying the kanamycin resistance marker. This initial culture has been grown across a series of liquid and solid LB kanamycin media containing increasing concentrations of NaCl salt. All salt concentrations reported indicate the amount in addition to that already present in the LB media used. All LB media has been made from the same Difco LB Broth, Lennox, powder lot. All liquid growths are considered ready for the next round of dilution once the broth has reached a turbidity that is easily identified with visual inspection. When transferring liquid growths onto solid media, different concentrations are plated and the concentration that yields individual colonies on the agar plate is used. Only completely resolved colonies are picked to undergo further selection. -80C stocks are made by adding glycerol to the fully grown culture for a final concentration of 30%. Characterization of the directed evolution assay progress is carried out with a Bioscreen C Growth Curves Reader (Transgalatic Ltd.). Samples are revived from -80 stocks or picked from plates into 5 ml overnight cultures. These overnights are then used to inoculate 200μL cultures that are grown at 37C (with vigorous shaking) and analyzed for wideband optical density in an automated fashion by the Bioscreen machine. Figure Explanations: Figure 1 shows the growth curves generated by the Bioscreen C instrument of 200uL samples inoculated with 1uL of overnight culture. All samples were grown concurrently in the machine at 37C while being shaken vigorously. Optical density across 420-580nm was measured at 15 minute intervals. All samples were analyzed in triplicate with the average curve displayed and errors bars indicating the standard deviation (exception -- the red curve on the 3.0M graph is the average of only two samples). The colors of the curves correspond to their respective fill color in the dilution series shown in Figure 2. Brown lines indicate non-inoculated salt + LB kan negative controls. Figure 1: Growth Curves Figure 2: Dilution Series Figure 2 shows the dilution series that has been carried out for this experiment. The shapes are explained in the key at right. The amount of NaCl salt added to LB broth is indicated below each growth. The amount of material transferred between growths is indicated over the arrow between them. If nothing is indicated over the arrow, then multiple concentrations of the liquid culture were plated to find individual colonies. The colored growths indicate those that were cultured for characterization and correspond with the colored curves in Figure 1.

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