Difference between revisions of "Part:BBa K530000"

Revision as of 16:24, 16 October 2011

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

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.

@@ Line 18: / Line 18: @@
 [[Image:CrtYB Plasmid Map.png]]
-===mCherry Tagging===
+===Localization of crtYB-mCherry and beta-carotene in yeast===
-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.
+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.
 [[Image:MCherry tagging of CRTYB.png]]