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| − | + | <h2 class="h2style">Integration of Olfr1258 and Ri7</h2> | |
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For the biosensor to work, modifications of the two yeast strains, a and α, needed to be performed. One major modification was the substitution of the native GPCRs, Ste2 and Ste3, with the two heterogenous olfactory receptors, Ri7 and Olfr1258. This was executed using a CRISPR system with Cas9 and gRNA specific to STE2 and STE3, for more information see <a href="https://2017.igem.org/Team:Chalmers-Gothenburg/Project/constructs" target="blank">Project constructs</a>. | For the biosensor to work, modifications of the two yeast strains, a and α, needed to be performed. One major modification was the substitution of the native GPCRs, Ste2 and Ste3, with the two heterogenous olfactory receptors, Ri7 and Olfr1258. This was executed using a CRISPR system with Cas9 and gRNA specific to STE2 and STE3, for more information see <a href="https://2017.igem.org/Team:Chalmers-Gothenburg/Project/constructs" target="blank">Project constructs</a>. | ||
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| + | By using colony PCR, the substitution/integration of the GPCRs could be validated, after transformation of sequences for GPCR and CRISPR system. The colony PCRs showed successful results for the incorporation of both of the GPCRs into the genome of the respective yeast strain. Olfr1258 has been incorporated into CENPK_111-61A and Ri7 into CENPK_11-11C. | ||
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| + | <h2 class="h2style">Membrane localization</h2> | ||
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| − | + | To enable visualization of the localization of the receptors in the yeast cells, the yeast was transformed with a plasmid containing the GPCRs fused together with GFP. The green fluorescence could then easily enable detection of the GPCRs. For the yeast strain with Olfr1258, the membrane localization of the GPCR could be verified. In the first figure, it can be seen that Olfr1258 is clearly located in the outer membrane of the cell. In the second figure, it can be seen that these cells fluoresce in the nuclear membrane which is clearly shown when the cells in this image is dividing. This image thereby suggest that the GPCR is localized in all membranes. | |
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| + | <div><b>Figure 1.</b></div> | ||
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| − | + | For the yeast cells expressing Ri7 fused together with GFP it could be visualized that this GPCR is membrane-localized as well. | |
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Revision as of 17:43, 25 October 2017
Overview
The lung cancer biosensor was divided into two major parts: receptor integration and pathway. The native membrane localized GPCRs STE2 and STE3 were substituted with the two heterologous GPCRs Olf1258 and Ri7 in two different yeast mating types. These two heterologous GPCRs were fused with GFP to test membrane localization. Finally, the functionality of the GPCRs was tested using the plasmid pGFP, were pFUS1 is coupled to GFP. pFUS1 is a mating gene promoter induced after GPCR induction. For testing the functional pathway, the loxP-pTEF-loxP switch was investigated. Before the pTEF promoter is turned, GFP is expressed. Therefore, the decrease in GFP expression was tracked after the turning of the promoter, indicating that the promoter switch was successful.
Receptors
Integration of Olfr1258 and Ri7
For the biosensor to work, modifications of the two yeast strains, a and α, needed to be performed. One major modification was the substitution of the native GPCRs, Ste2 and Ste3, with the two heterogenous olfactory receptors, Ri7 and Olfr1258. This was executed using a CRISPR system with Cas9 and gRNA specific to STE2 and STE3, for more information see Project constructs.
By using colony PCR, the substitution/integration of the GPCRs could be validated, after transformation of sequences for GPCR and CRISPR system. The colony PCRs showed successful results for the incorporation of both of the GPCRs into the genome of the respective yeast strain. Olfr1258 has been incorporated into CENPK_111-61A and Ri7 into CENPK_11-11C.
Membrane localization
To enable visualization of the localization of the receptors in the yeast cells, the yeast was transformed with a plasmid containing the GPCRs fused together with GFP. The green fluorescence could then easily enable detection of the GPCRs. For the yeast strain with Olfr1258, the membrane localization of the GPCR could be verified. In the first figure, it can be seen that Olfr1258 is clearly located in the outer membrane of the cell. In the second figure, it can be seen that these cells fluoresce in the nuclear membrane which is clearly shown when the cells in this image is dividing. This image thereby suggest that the GPCR is localized in all membranes.
For the yeast cells expressing Ri7 fused together with GFP it could be visualized that this GPCR is membrane-localized as well.
