Difference between revisions of "Part:BBa K1728024"
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The rhodopsin-CXCR1 sequence is a combination of | The rhodopsin-CXCR1 sequence is a combination of | ||
| − | 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) |
| − | + | ==Construction of new yeast integrating plasmids== | |
| + | Plasmids cloning vectors that can “shuttle” DNA between yeast and bacteria we used is called pGAL426. Distinctly, it has different selection markers in yeast and bacteria. Rho-CXCR1 was inserted into this shuttle vector and finally transformed into FAR1△::KanMX-FUS1-GFP Gpa1 chimera strain. | ||
| + | https://static.igem.org/mediawiki/2015/d/d0/CGU-pGAL426_rho-CXCR1_with_BamHI_and_XhoI_digestion.jpg | ||
| − | + | The result of pGAL426 rho-CXCR1 with BamHI and XhoI digestion. | |
| + | Note: These two restriction enzyme site designed on insert sequence was used to check the ligation performance by enzyme digestion. Theoretically, there would be two bands. | ||
| − | |||
| − | + | ==Western blot analysis of Rho-CXCR1 expression== | |
| − | https://static.igem.org/mediawiki/2015/7/ | + | https://static.igem.org/mediawiki/2015/7/71/Figure_5-1_Western_blot_analysis_of_rho-CXCR1_expression.jpg |
| + | |||
| + | 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/2015/9/93/Final_data_figure.jpg | ||
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. | ||
Latest revision as of 09:39, 18 June 2016
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)
Construction of new yeast integrating plasmids
Plasmids cloning vectors that can “shuttle” DNA between yeast and bacteria we used is called pGAL426. Distinctly, it has different selection markers in yeast and bacteria. Rho-CXCR1 was inserted into this shuttle vector and finally transformed into FAR1△::KanMX-FUS1-GFP Gpa1 chimera strain.
The result of pGAL426 rho-CXCR1 with BamHI and XhoI digestion. Note: These two restriction enzyme site designed on insert sequence was used to check the ligation performance by enzyme digestion. Theoretically, there would be two bands.
Western blot analysis of Rho-CXCR1 expression
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
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