Composite

Part:BBa_K1728024

Designed by: Wan-Yun Chiu, Timothy Chan En Haw, Ming-Fei Hsieh   Group: iGEM15_CGU_Taiwan   (2015-09-17)
Revision as of 03:55, 19 September 2015 by Wanyun (Talk | contribs) (Usage and Biology)

rhodopsin siganl peptide +IL-8 receptor type alpha (CXCR1)

rhodopsin siganl peptide +IL-8 receptor type alpha (CXCR1)

Usage and Biology

The rhodopsin-CXCR1 sequence is a combination of bovine rhodopsin signal peptide (BBa_K1728020) and CXCR1 with optimized sequence(BBa_K1728021)

Western blot analysis of Rho-CXCR1 expression

File:Https://static.igem.org/mediawiki/2015/4/4a/CGU Taiwan r08.tif

The galactose induction of CXCR1 expression was performed as described in protocol. We collect yeast samples at different induction time. Protein extracted from the yeast whole-cell extract was separated on SDS-PAGE and finally detected by anti-CXCR1 antibody.

Effect of IL-8 treatment in engineered yeast with Rho-CXCR1 expression

File:Https://static.igem.org/mediawiki/2015/4/4a/CGU Taiwan r08.tif

Plasmid p426GAL-CXCR1 was transformed into engineered yeast, and CXCR1 was induced by 2 % galactose overnight. At first, we could not see GFP production. In order to exclude the possibility that cell wall blocks the binding between CXCR1 and IL-8, yeast cells were digested with zymolyase to remove cell wall and became spheroplasts. After that, we add 1 μM IL-8 to stimulate GPCR-mediated pathway, we observed GFP after IL-8 treatment for 8 hours. The negative control is the same yeast strain without IL-8 treatment. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 852
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 277
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 49
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1041


[edit]
Categories
//chassis/eukaryote/yeast
Parameters
None