Phase II

Design

The purpose of phase II is to optimize the expression of the proteins necessary for the production of crocin glycosides and safranal. Unfortunately, ZCD has been shown to produce inclusion bodies when expressed'' in vivo'' in ''E. coli'' and so an alternative method needed to be developed to create a functional protein. This was done by following the technique described by Florence Bouvier (The Plant Cell, vol. 15 47-62).

Results

The soluble protein in the protein which was transformed with the tagged pro was extracted and analyzed by SDS-PAGE gel electrophoresis. The result showed showed a protein of the target mass that was found primarily in the induced cells. This result was further confirmed by digestion of the band in question using trypsin (Sigma Aldrich, St. Louis) and analyzed using LC-MS (Waters Synapt G2 and HPLC).


The information that lead to the development of the this vector tag with ZCD came out of the following paper: Bouvier, Florence et al. “Oxidative Remodeling of Chromoplast Carotenoids : Identification of the Carotenoid Dioxygenase CsCCD and CsZCD Genes Involved in Crocus Secondary Metabolite Biogenesis.” 15.January (2003): 47–62.