Part:BBa_K1343016:Experience

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Applications of BBa_K1343016

To test the activity of the Taz construct we created, varying concentrations of L-aspartic acid were added to a culture of E. coli expressing the Taz construct on plasmid pSB1AK3 and RFP under the promoter PompC (biobrick Bba_M30011 designed by Natalie Kuldell) on plasmid pSB1C3. The experiment was based on Dundee iGEM 2013 team’s experiment with ompC-GFP reporter construct (BBa_K1012005 designed by John Allan) (Harrison, 2013) and the research of Michalodimitrakis, Sourjik and Serrano who tested Taz expression using GFP under PompC regulation (Michalodimitrakis, Sourjik, & Serrano, 2005) (See Figure 1)

[File:Taz-GFP.png‎]

Figure 1: The chimeric Taz-ompR System as tested by Michalodimitrakis, Sourjik and Serrano (Michalodimitrakis, Sourjik, & Serrano, 2005)

Two isogenic strains of E. coli K-12, BW25113 (parent strain for the Keio collection) and JW3367-3 (with ΔEnvZ mutation) were transformed with pSB1AK3 carrying our Taz construct (BBa_K1343016) and pSB1C3 carrying the BBa_M30011 reporter. The bacteria were cultured in growth media containing varying concentrations of L-aspartic acid. After two hours of growth the relative RFP fluorescence of the cultures was determined (fluorescence/OD).

OD was measured at 600nm. Fluorescence excitation wavelength: 560nm Fluorescence emission wavelength: 612nm

[File:Taz_result.jpg] Figure 5: Relative fluorescence dependent on L-aspartic acid concentration (mM)

Figure 5 shows that at low concentrations of L-aspartatic acid (below 1mM), there is a steady, constant expression of the reporter in both the wild type and the mutant strain. The relative fluorescence observed by the mutant strain (ΔEnvZ) is lower than that of the parent strain. This is because the natural histidine kinase protein which detects a high level of osmolarity in the cells environment (EnvZ) is present in the wild type. This contributes to phosphorylation of the ompR. The low level of fluorescence observed in the mutant strain could be due to another mechanism (such as an acetyl phosphate dependent mechanism) which phosphorylates the ompR, leading to activation of the PompC promoter.

The mutant shows an increase in relative fluorescence is observed at 1.33mM L-aspartic acid, with a peak at 2mM followed by a drop in relative fluorescence at 4mM. The drop in relative fluorescence in both the mutant and wild type indicates a toxic concentration level.

In the mutant strain, the relative fluorescence increases 202% in comparison to the basal level. This occurs over a narrow concentration range, which reflects the sensitivity of the two-component signaling system.

References

Baba, T., Ara, T., Hasegawa, M., Takai, Y., & Okumura, Y. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology, 1-11. Forst, S. A., & Roberts, D. L. (1994). Signal transduction by the EnvZ-OmpR phosphotransfer system in bacteria. Research in Microbiology, 145, 363-373. Harrison, K. (2013). Reporter ompC-GFP. Retrieved from Toximop: http://2013.igem.org/Team:Dundee/Project/ReporterOmpC Heyde, M., Laloi, P., & Portalier, R. (2000). Involvement of Carbon Source and Acetyl Phosphate in the External-pH-Dependent Expression of Porin Genes in Escherichia coli. Journal of Bacteriology, 182(1), 198-202. Levskaya, A., Chevalier, A. A., & Tabor, J. J. (2005). Engineering Escherichia Coli to see light. Nature, 438(24), 441-442. Michalodimitrakis, K. M., Sourjik, V., & Serrano, L. (2005). Plasticity in amino acid sensing of the chimeric receptor Taz. Molecular Microbiology, 58(1), 257–266. Tabor, J. J., Groban, E. S., & Voigt, C. A. (2009). Performance Characteristics for Sensors and Circuits Used to Program E. coli. In S. Y. Lee (Ed.), Systems Biology and Biotechnology of Escherichia coli (pp. 401-439). Springer Science+Business Media B.V. Yoshida, T., Phadtare, S., & Inouye, M. (2007). The Design and Development of Tar-EnvZ Chimeric Receptors. Methods in Enzymology, 423, 166-183.

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