Team:Missouri Rolla/Parts

Missouri cave photographed by Lynn Dieter

PARTS

Part BBa_K1737000 is an improvement of last year's BBa_K1370000, nitric oxide dioxygenase from the hmp gene of E. coli. We added the promoter and ribosome binding site BBa_K608002 to the coding region. We also prepared our part for GST-tagging and purification, but were unable to finish before Jamboree.

The remainder of our parts for this year's project were designed for Gibson assembly and ordered in short fragments as IDT gBlocks. Unfortunately, our Gibson assemblies failed, most likely due to secondary structure in overlap regions. We are now ordering the same genes for Standard Assembly, but will have to finish and submit the planned parts after Jamboree. Parts 1 and 2 together form the mevalonate pathway after assembly, and produce (E)-beta-ocimene from Acetyl-CoA. Part 3 is isolated ocimene synthase for use in studying enzymatic characteristics.

Part 1 includes the mvaE and mvaS genes from Enterococcus faecalis. The enzyme coded by mvaE performs two distinct steps in the mevalonate pathway.

  • Sequence (four fragments)
  • Yoon, S. H., Lee, S. H., Das, A., Ryu, H. K., Jang, H. J., Kim, J. Y., ... & Kim, S. W. (2009). Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli. Journal of biotechnology, 140(3), 218-226.

Part 2 features MVK from Methanosarcina mazei, EF_0902 from E. faecalis, MVD1 from Saccharomyces cerevisiae, idi from E. coli, ERG20-2 from Part BBa_K517005, and TPS02 from Arabidopsis thaliana.

  • Sequence 1/2 (four fragments)
  • Sequence 2/2 (four fragments)
  • Beck, Z. Q., Calabria, A. R., Miller, M. C., Vaviline, D. V., Nielsen, A. T. (2013). Increased isoprene production using the archaeal lower mevalonate pathway. US Patent 8361762 B2.
  • Hahn, F. M., Hurlburt, A. P., & Poulter, C. D. (1999). Escherichia coli open reading frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate isomerase. Journal of bacteriology, 181(15), 4499-4504.
  • Primak, Y. A., Du, M., Miller, M. C., Wells, D. H., Nielsen, A. T., Weyler, W., & Beck, Z. Q. (2011). Characterization of a feedback-resistant mevalonate kinase from the archaeon Methanosarcina mazei. Applied and environmental microbiology, 77(21), 7772-7778.

Part 3 is ocimene synthase (TPS02) from A. thaliana, which produces the final product, (E)-beta-ocimene.

  • Sequence (including VR-complementary and VF2 sites for amplification)

Missouri cave photo by Lynn Dieter