Difference between revisions of "Part:BBa K1728024"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1728024 short</partinfo>
 
<partinfo>BBa_K1728024 short</partinfo>
  
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rhodopsin+Chemokine receptor 1 (CXCR1)
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rhodopsin siganl peptide +IL-8 receptor type alpha (CXCR1)
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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The rhodopsin-CXCR1 sequence is a combination of
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bovine rhodopsin signal peptide (BBa_K1728020) and CXCR1 with optimized sequence(BBa_K1728021)
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Western blot analysis of Rho-CXCR1 expression
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https://static.igem.org/mediawiki/2015/4/4a/CGU_Taiwan_r08.tif
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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.
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Effect of IL-8 treatment in engineered yeast with Rho-CXCR1 expression
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https://static.igem.org/mediawiki/2015/7/74/CGU_Taiwan_r10.tif
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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.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 03:54, 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/2015/7/74/CGU_Taiwan_r10.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