"
Team:HKUST/Group1
From 2009.igem.org
a
Construction of Receptor Expression Cassette
To functionally express C. elegans ODR-10 receptor, we have designed an expression cassette. Based on the fact that rat I7 (RI7) receptor has been successfully expressed in the yeast system with effective coupling to the Gαolf subunit, we have designed a chimeric receptor with the RI7 N (amino acids 1-61 of RI7) and C termini (animo acids 295-327 of RI7) fused to the ODR-10 transmambrane domains two to seven (TM2 – 7, amino acids 48-305 of ODR-10, starting from the fourth amino acid of Odr10 TM2) [7,8] (Figure 3). At the N terminus side, the two fragments are fused at the junctions PMYFF (RI7) and YLMAFF (ODR-10), because these two sequences are conserved in both receptor junction sites (Figure 4).The fused chimeric receptor is then cloned into the multiple cloning site of pESC-HIS expression vector under the GAL1 promoter. It is also tagged with FLAG or GFP at the C terminus for protein expression detection or localization test. Given that we have retained the N and C termini of RI7, localization and subsequent coupling should not be perturbed.
Test of Chimeric Protein Localization
To test the localization of chimeric receptors, we have made the construct pESC-Fusion-GFP, which is the chimeric protein with the GFP tag at the C terminus in the expression vector pESC-HIS, as well as the negative control pESC-GFP, which has only GFP cloned into the same site in the expression vector pESC-HIS (Figure 5). We choose to tag GFP at the C terminus because the N terminus of the fusion protein is important for membrane localization while the C terminus is important for downstream coupling. Since we want to ensure proper localization of the protein, it is better to fuse GFP at the C terminus.
After transformation and selection, we would induce the transformants with galactose at the exponential stage of yeast growth. After some time, we would harvest cells for the fluorescence microscopy test. We would expect to see the cells transformed with pESC-Fusion-GFP to have strong green fluorescence surrounding the cell, forming a green halo; whilst the negative control does not. In that case, we could say that the fusion protein is able to localize in the yeast membrane.
Test of Chimeric Protein Function
To test the functional odorant sensing and Gα subunit coupling, we have made several constructs:
There are two parts in the fuctional coupling test:
Part 1
First we test whether our chimeric protein could couple to Gpa1.
The yeasts would be transformed with construct (1)+(4) or (2)+(4) or (5)+(4), respectively. After induction with galactose to express the receptors, we would add the ligands diacetyl (for ODR-10) and hexanal (for RI7). After some time of ligand binding, we would expect to see that the functional receptors could couple to Gpa1 and then trigger downstream FUS1-promoter-driven expression of GFP, together with cell cycle arrest at G1 phase (figure 6). The expression of GFP could be viewed by fluorescence microscopy; the cell cycle arrest could be confirmed by fluorescence-activated cell sorting (FACS). We expect to see the fusion receptor and RI7 respond to diacetyl and hexanal respectively, and have GFP expression and cell cycle arrest. In that case we could say that fusion receptor could functionally couple to Gpa1 and initiate downstream signalling.
Part 2
Second, we test whether our chimeric receptor could couple to Gαolf for optimized signal transduction, as well as construct a testable yeast system for odorant sensing.
Before testing, the yeast strain needs to be manipulated: we need to knock out the FAR1 gene, which encodes a protein that controls cell cycle arrest upon activation of the MAPK pathway. After deletion of the FAR1 gene, the yeast will still be viable after ligand binding. We also need to knock out the endogenous GPA1 gene, so that we could replace it with Gαolf. Due to the second messenger activity of the free G protein βγ subunit, haploid S. cerevisiae strains with a null mutation of GPA1 undergo constitutive pheromone response of G1 arrest, resulting in non-viability[9]. So, we need to first knock out the cell cycle regulatory gene FAR1 and afterwards, GPA1. To knock out these two genes, we have adopted the PCR-based tagging of yeast genes method, introduced by Janke C., et al, 2004[10].
To test for successful deletion, we will use both PCR confirmation (Figure 7) and phenotype observation after adding α-factor (no mating phenotype).
Next, we need to transform Gαolf into the double knock-out strain to check for positive transformants. It has been reported that Gαolf can not only complement for a GPA1 deficiency but also functionally couple to the pheromone receptor STE211. Thus we have developed assays to select for functional Gαolf transformants (Figure 8).
After confirmation of functional Gαolf, we would use this strain to test its ligand binding using the same method shown in Figure 6. However, this time we would expect to see no cell cycle arrest in any case since FAR1 has been deleted. We expect the ligand binding assay this time to give an optimized coupling and signal transduction.
Further Characterization of the Chimeric Protein Function
To further characterize how this chimeric receptor functions, we can do the following assays:First, we can test how the recptor responds to different ligand concentrations. According to literature, C. elegans ODR-10 gives strongest response to 100μM diacetyl[11], and we can then compare the intensity of GFP production under a series of diacetyl concentrations.
Second, we can test the ligand specificity of the chimeric protein. According to literature, ODR-10 responds to the volatile molecule diacetyl, as well as the non-volatile molecules pyruvic acid and citric acid[13]. We can also test whether it can respond to other volatile molecules by setting up similar assays as shown in Figure 6.
iGEM 2009
