Difference between revisions of "Part:BBa K1728024"
(→Usage and Biology) |
(→Usage and Biology) |
||
| Line 9: | Line 9: | ||
bovine rhodopsin signal peptide (BBa_K1728020) and CXCR1 with optimized sequence(BBa_K1728021) | bovine rhodopsin signal peptide (BBa_K1728020) and CXCR1 with optimized sequence(BBa_K1728021) | ||
| − | Western blot analysis of Rho-CXCR1 expression | + | ==Western blot analysis of Rho-CXCR1 expression== |
| − | + | 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. | 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 | + | ==Effect of IL-8 treatment in engineered yeast with Rho-CXCR1 expression== |
| − | + | 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. | 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. | ||
Revision as of 03:58, 19 September 2015
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
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
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 852
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 277
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 49
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1041