Part:BBa_K771303:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K771303

We codon optimized and characterized BBa_K771303 for expression in E. coli with the pET29b+ plasmid. The protein sequences are the same, but the nucleotides were codon optimized, see below for the alignment.

BBa_K771303 and BBa_K1712000 Nucleotide Alignment:

This new part can be found here: https://parts.igem.org/Part:BBa_K1712000

Usage and Biology of BBa_K1712000

Protein purity assessed through SDS-PAGE gels shown below: Enzyme Expression Assay:

Enzyme Activity Assay:

Triclosan Inhibition Screening

Since Chalew et al showed the levels of triclosan leaving Waste Water Treatment Plants (WWTPs) was up to 9 nanomolar [18], we wanted to measure enzyme inhibition using a nanomolar level of triclosan. Under our conditions, however, not all of the fabI enzymes had measurable activity with a nanomolar amount of enzyme, and in order to see inhibition using a nanomolar amount of triclosan we needed to use a nanomolar amount of enzyme.

Enzyme Inhibition Assay:

Triclosan inhibition was measured by running our standard enzyme activity assay with no triclosan and 1 nM triclosan. Negative control was 100 uM NADH, 100 uM crotonyl CoA, no enzyme, no triclosan. Observed enzyme activities were subtracted from negative control activities. Percent inhibition was calculated by:

( (uninhibited activity - inhibited activity) / uninhibited activity ) * 100

References

[3] McMurry, Laura M., Margret Oethinger, and Stuart B. Levy. "Triclosan Targets Lipid Synthesis." Nature 394 (1998): 531-32. Web. [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): n. pag. Web. [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): n. pag. Web. [7] Ward, Walter. "Kinetic and Structural Characteristics of the Inhibition of Enoyl (acyl Carrier Protein) Reductase by Triclosan." Biochemistry (1999 Sep 21): n. pag. Web. [8] Kapoor, Mili. "Slow-tight-binding Inhibition of Enoyl-acyl Carrier Protein Reductase from Plasmodium Falciparum by Triclosan." Biochem (2004 August 1): n. pag. Web. [10] Marcinkeviciene, J.et 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. [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 [23]. Mol. Biol. (2004) 343, 147–155 doi:10.1016/j.jmb.2004.08.033 [29] Triclosan causes toxic effects to algae in marine biofilms, but does not inhibit the metabolic activity of marine biofilm bacteria Marine Pollution Bulletin 84 (2014) 208–212

User Reviews

UNIQ08833fefeba866a3-partinfo-00000000-QINU UNIQ08833fefeba866a3-partinfo-00000001-QINU