Part:BBa_K530000

CRTYB

Enzyme in the pathway required for B-Carotene Synthesis. This enzyme is a combination, it is the full Phytoene Synthase enzyme spliced with a Lycopene B-Cyclase enzymatic domain. This sequence was taken from a WT strain of xanthophyllomyces dendrorhous. It catalyzes both the conversion of Geranylgeranyl diphosphate to Phytoene and Lycopene to B-Carotene.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1068
  Illegal BsaI.rc site found at 1399
  

Plasmid Map

Characterization of performance

Below are plotted the beta-carotene production levels on a per cell basis for different combinations of parts involved in beta-carotene synthesis. These are compared with a wild type control (WT). tHMG1 is used to funnel more initial substrate into the pathway and although useful, was not submitted as a part. 2I indicates that two copies of crtI were used.

Localization of crtYB-mCherry and beta-carotene in yeast

Yeast cells, previously engineered to express all genes in the beta carotene biosynthetic pathway were subsequently transformed with a copy of crtYB C-terminally tagged with mCherry. The subcellular localization pattern of mCherry (crtYB-mCherry) was then assessed by fluorescence microscopy as compared to the parental, untagged control strain (crtYB) under identical conditions. Autofluorescence of beta carotene using a filter set for fluorescein fluorescence was also performed. Yeast cells are shown using differential interference contrast (DIC). Microscopy was performed at 1000X magnification.

Sequencing

This is the sequencing for colony 2. Colony 2 had areas of poor match between both forward and reverse regions. We attribute this to the fact that our gene is far too big for two primers to cover on their own. The middle regions are due to this, and the initial and final regions are due to the trace taking some time to start up.