"
Team:HKUST/Group1
From 2009.igem.org
a
Construction of Receptor Expression Cassette
To functionally express C. elegans Odr10 receptor, we have designed an expression cassette. Based on the fact that rat I7 (RI7) receptor has been successfully expressed in the yeast system and it couples well to Gαolf subunit, we have designed such a chimeric receptor with the RI7 N (amino acids 1-61 of RI7) and C termini (animo acids 295-327 of RI7) fused with Odr10 transmambrane domains two to seven (TM2 – 7, amino acids 48-305 of Odr10, starting from the fourth amino acid of Odr10 TM2) [i] (figure 3). At the N termini side, they are fused at the junctions PMYFF (RI7) and YLMAFF (Odr10) because these two sequences are conserved in both receptor junction sites (Figure 4). They are cloned into the multiple cloning site of pESC-HIS expression vector under the Gal1 promoter. They are also tagged with FLAG or GFP at the C termini 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.
Fig3. Schemetic illustration of receptor expression strategy. The receptor expression cassette of the pESC-HIS vector was constructed to contain an insert that encodes the N and C termini of the RI7 receptor flanking an intervening sequence containing multiple cloning sites. The ligand-binding pocket of Odr10 protein is inserted between N and C termini. Adopted from Venkat,B.,el al, 2007. Fig 4. Sequence of the chimeric receptor. Transmambrane domains are highlighted in red;sequences from RI7 are highlight in yellow; the fusion site is separated by a green line.
Test of Chimeric Protein Localization
To test the localization of chimeric proteins, we have made the construct pESC-Fusion-GFP, which is the chimeric protein with the GFP tag at the C termini in the expression vector pESC-HIS, as well as the negative control pESC-GFP, which is only the GFP at the same site in the expression vector pESC-HIS (Figure 5). We choose to tag GFP at the C termini because N termini of the fusion protein is important for membrane localization while C termini is important for coupling. Since we want to ensure its proper localization, it is better to fuse GFP at the C termini.
Fig 5. Constructs for chimeric receptor fluorescence miscroscopy test, in order to confirm its localization to the yeast membrane. (a) is pESC-Fusion-GFP, which is the chimeric protein with the GFP tag at the C termini in the expression vector pESC-HIS; (b) is the negative control pESC-GFP, which is only the GFP at the same site in the expression vector pESC-HIS. 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 circle; while 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 sensing and coupling to Gαolf or Gpa1, we have made several constructs:(1) pESC-Fusion-FLAG, which is the chimeric protein with the FLAG tag at the C termini in the expression vector pESC-HIS under the Gal1 promotor.
(2) pESC-RI7-FLAG, which is the rat RI7 receptor protein with the FLAG tag at the C termini in the expression vector pESC-HIS under the Gal1 promotor, serving as the positive control.
(3) pESC-Gαolf-FLAG , which is rat Gαolf in the pESC-HIS under the Gal10 promotor.
(4) pRS426-FUS1P-GFP-FUS1T, which is the FUS1 promotor, GFP and FUS1 terminator in the expression vector pRS426.
(5) pESC empty plasmid, serving as the negative control. There are two parts of 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 in the ligands diacetyl (for Odr10) and hexanal (for RI7). After some time of ligands 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 might expect to see the fusion receptor and RI7 response to diacetyl and hexanal, respectively, to have GFP and cell cycle arrest. In that case we could say that fusion receptor could functionally couple to Gpa1 and start downstream signalling.
Fig 6 Ligands sensing functional assay showing control experiments. (a) is yeast transformed with pESC-Fusion-FLAG & pRS426-FUS1P-GFP-FUS1T, and we would expect to see GFP and cell cycle arrest only when both galactose and diacetyl are added; (b) is yeast transformed with pESC-RI7-FLAG & pRS426-FUS1P-GFP-FUS1T, and we would expect to see GFP and cell cycle arrest only when both galactose and hexanal are added; (c) is yeast transformed with pESC empty plasmid and pRS426-FUS1P-GFP-FUS1T, and we would expect to see no GFP and cell cycle arrest. These cells are analyzed with fluorescence microscopy and FACS. Part 2
Second, we test whether our chimeric receptor could couple to Gαolf for optimized signalling transduction as well as constructing a testable yeast system for odorant sensing.
Gene deletion
Before testing, yeast strain needs to be manipulated: we need to knock out the FAR1 gene, which encodes a protein controlling cell cycle arrest upon activation of MAPK pathway, so that the yeasts will still be viable after ligand binding; and also 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 containing a null mutation of GPA1 undergo constitutive pheromone response of G1 arrest resulting in non-viability[ii]. So, we need to first knock out 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., el al, 2004[iii].
To test the successful deletion, we will use both PCR confirmation (figure 7) and also phenotype observation after adding α-factor (no mating phenotype). Figure 7. Primers designed for colony PCR to confirm successful deletion of yeast ORF. The correct replacement of the gene with hphNT1 or natNT2 is verified in the mutants by the appearance of PCR products of the expected size using primers that span the left and right junctions of the deletion module within the genome. Next, we need to transform Gαolf into the double knock-out strain to check for positive transformants. It has been reported that Gαolf could not only complement for a GPA1 deficiency but also can functionally couple to the pheromone receptor STE211. Thus we have developed assays to select functional Gαolf (figure8). Fig 8. Assay testing the Gαolf functional expression in yeasts that have been knocked out with FAR1 and GPA1. In (a), yeasts are transformed with pESC-Gαolf & pRS426-FUS1P-GFP-FUS1T; in (b), yeasts are transformed with pESC empty vector & pRS426-FUS1P-GFP-FUS1T. Both are induced with galactose and then α factor, and afterwards cells are viewed with fluorescence microscopy. In (a) Gαolf could couple with STE2 and Gβγ subunits and hence triggers GFP production; negative transformants and control in (b) will not trigger GFP production. 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 could expect the ligand binding assay this time could give an optimized coupling and signalling transduction.
Further Characterization of the Chimeric Protein Function
To further characterize how this chimeric receptor function, we can do the following assays:First, we can test the how it responses to different ligand concentrations. According to literature, C. elegans Odr10 responses strongest to 100μM diacetyl[i], 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, Odr10 responses to volatile molecule diacetyl, as well as non-volatile molecule pyruvic acid and citric acid13. We can also test whether it can response to other volatile molecules by setting up similar assays shown in figure 6.
iGEM 2009
