Difference between revisions of "Part:BBa K1728024"

 
 
(7 intermediate revisions by 3 users not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1728024 short</partinfo>
 
<partinfo>BBa_K1728024 short</partinfo>
  
−
rhodopsin+Chemokine receptor 1 (CXCR1)
+
rhodopsin siganl peptide +IL-8 receptor type alpha (CXCR1)
  
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===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.
 +
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/2015/7/71/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.
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

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. 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

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

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. 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