Team:Chalmers-Gothenburg/Results

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Results Summary

Spot test analysis and growth measurements of yeast in urine were performed in order to test the survival of yeast cells in urinary medium. These initial experiments showed that yeast cells are able to survive in urinary medium which is an important prerequisite for the biosensor.

The gene CWP2, encoding a mannoprotein in the cell wall was successfully deleted in the yeast strain IMFD-73. The deletion was confirmed by verification PCR. In addition, a lyticase assay showed that [IMFD-73 Δcwp2::kanMX] was degraded faster than IMFD-73 and this leads us to the conclusion that the cell wall is somewhat weakened in the strains with the deleted cell wall mannoprotein CWP2.

Two different plasmids both containing the receptor gene LHCGR but one with the human signal peptide and the other one with a yeast signal peptide was created. Both plasmids were successfully cloned into IMFD-73 and [IMFD-73 Δcwp2::kanMX] but in neither strains could the receptor be proved as functional in detection of hCG.

The Indigo group managed to obtain different colour changes in different growth media by introducing a plasmid containing the genes tnaA and fmo (pIndigo). For instance, blue bubbles could be seen in one medium and other media turned into different shades of brown. These changes of colour were strictly dependent on the presence of pIndigo and could not be detected in any of the cultures with the strain containing the control plasmid. This indicates an at least partial activity of the pathway. However, we did not manage to confirm the presence of bio-indigo and to determine exactly which compound or reaction that was responsible for the colourful bubbles or colour changes.

[IMFD-73 Δcwp2::kanMX] was successfully transformed with both receptor genes and genes required for bio-indigo production. However, the system was never functional as a pregnancy test kit since it did not give any significant response in the presence of hCG. More detailed analysis and the discussion of the results and discussion can be found in the sections below.

Survival of yeast in urine

Figure 1: Growth curve for cells in urine and YPD medium. The OD remains the same in the urine i.e. the cells are not growing.
An initial test was performed in order to test the survival of yeast cells in urine, which is an important property of our biosensor if it should function as a pregnancy test kit. Pre-cultures of the lab yeast strain CEN.PK 113-11C were prepared in 5 ml YPD and grown O/N at 30 °C. The day after, the cells were centrifuged, resuspended in 40 ml YPD and 40 ml filter sterilized urine respectively and grown in shake flasks at 30°C. OD600 measurements were taken at two different points of time during the day. The growth curve can be seen in Figure 1. The cells in urine did not grow; the OD remained the same during the whole
Figure 2: Spot test analysis of cells grown in YPD and urine for 4h. The cells survive in the urine and grow normal after being plated on a YPD plate.
day, while the cells in YPD were proliferating.


In order to test if the cells remained alive in the urin, 300 µl of each culture (after 4h) were taken and OD600 was adjusted to 0.5. The samples were diluted 3x, 9x, 27x and 81x and 10 µl aliquots of each dilution were spotted on an YPD plate (Figure 2). The cells from both the YPD and the urine media could grow, this means that the cells survived 4 h in urine and grew normal after being spotted on a YPD plate. Summarizing, yeast cells are unable to proliferate in the urine medium but do survive under these conditions.

Deletion of CWP2 gene

One task of our iGEM project was the deletion of the CWP2 gene, which is encoding a cell wall mannoprotein. By removing it, we aimed for higher cell wall permeability and thus enhanced chances of our ligand hCG to pass the cell wall and to bind to the membrane-bound receptor.

Gene Deletion according to the Bipartite method

The gene deletion was performed according to the bipartite method. The results from the first PCR reactions, in which we amplified the overlapping fragments, can be seen in Figure 3.

Figure 3: A) A schematic illustration of the first four PCR reactions. The 5' and 3' end of the kanMX (kanamycin resistance) cassette, flanked to loxP sites and available in vector pUG6h, were amplified by PCR (PCR reaction 1 and 2). In addition, 500 bp of the up- and downstream sequence of the CWP2 gene were amplified from genomic DNA using primers with 5' extensions that are complementary to the loxP sites as indicated with lines above. B) The results of the four PCR reactions on gel. The expected sizes of the kanMX and CWP2 fragments are about 1000 bp and 500 bp respectively which correspond to the sizes observed on gel.

The next step was to fuse together the overlapping fragments. The results of the fusion PCR are shown in the Figure 4. By transforming yeast with these fragments, homologous recombination should occur which will lead to the exchange of the CWP2 gene with the kanMX cassette.

Figure 4: A) A schematic illustration of the fusion PCR, the second step in the bipartite method. The CWP2 upstream fragment was fused with the 5’ end of the kanMX cassette and the CWP2 downstream fragment was fused with the 3’ end of the kanMX cassette. B) The results of the fusion PCR on gel. Two samples (A and B) of each PCR reaction are shown. The expected size of both fusion fragments is about 1500 bp which corresponds to the sizes observed on gel (indicated with an arrow).

Colony PCR

Yeast strain IMFD-73 was transformed with 150 ng of each fragment and grown on G418 plates. Colonies could be observed, which were purified, and colony PCR was then performed in order to test whether the kanMX cassette was integrated randomly in the genome or had replaced the CWP2 gene as desired. PCR was run with a forward primer outside of the inserted region and reverse primers within the CWP2 gene or kanMX cassette respectively. As a control, a wild-type strain and a Δcwp2 strain from a deletion library were also tested. The results of the PCR reactions indicated that the deletion was correct (see Figure 5).

Figure 5: A) Schematic illustration of the colony PCR. A forward primer was designed to bind upstream in the genomic DNA of the 500 bp region that had been used in the construction of the bipartite fragment. Two reverse primers were designed to hybridize within the CWP2 gene and in the kanMX cassette respectively. The primers are indicated with arrows. B) The results of the PCR reactions on gel. In every reaction, the cwp2-fw primer but different reverse primers were used. 1: The Δcwp2 deletion strain (bipartite) with the kanMX-rev primer. 2: The Δcwp2 deletion strain (from deletion library) with the kanMX-rev primer. 3: The Δcwp2 deletion strain (bipartite) with the cwp2-rev primer. 4: The Δcwp2 deletion strain (from deletion library) with the cwp2-rev primer. 5. A wild type yeast strain with the cwp2-rev primer. Since the CWP2 gene is missing and the kanMX cassette is inserted instead, bands are expected when PCR is run with the kanMX-rev primer (1, 2, expected size 1800 bp) but no bands should be observed when PCR is performed with the cwp2-rev primer (3, 4). On the contrary, a clear band of about 800 bp should be observed when running PCR with the wild type strain and the cwp2-rev primer. This corresponds to the result on gel which indicates that the CWP2 deletion was successful.

Lyticase assay

A lyticase assay was performed primarily in order to check the activity of lyticase. Another goal was to compare the rapidity of the cell wall degradation between the IMFD-73 and the [IMFD-73 Δcwp2::kanMX] strain. Lyticase was added to the cells and the cell wall degradation (i.e. protoplast formation) could be displayed by adding SDS and then measuring OD at 600 nm (SDS leads to lysis of protoplasts, while cells with intact cell wall remain unaffected). SDS was added and OD values were taken at different time points. The two graphs (Figure 6 and 7) below show the decrease of the amount of intact cells in % over a time period of 1h with two different amounts of lyticase. The percentages of intact cells were calculated as OD600(t=x)/OD600(t=0)*100. One can observe that a slightly smaller amount of cells of the deletion strain were intact between 30 and 60 minutes (except for one outlier at 50 min) which means that the cell wall degraded quicker in the [IMFD-73 Δcwp2::kanMX] strain. This lead to the conclusion that the cell wall is somewhat weakened in the strains with the deleted cell wall mannoprotein gene CWP2.

Figure 4: Assessment of cell wall degradation by lyticase (50 µl enzyme solution). Prior to the each OD measurement, SDS was added. The percentage of intact cells was calculated as OD600(t=x)/OD600(t=0)*100.
Figure 5: Assessment of cell wall degradation by lyticase (20 µl enzyme solution). Prior to the each OD measurement, SDS was added. The percentage of intact cells was calculated as OD600(t=x)/OD600(t=0)*100.

Introduction of genes for indigo-synthesizing enzymes

Expression of the gene for the LH/CG receptor in yeast

Two different versions of the LH/CG receptor were to be evaluated and expressed in yeast during this project: one with the original human signal peptide fused to the receptor and one with the yeast Ste3 signal peptide fused to the receptor. Both versions were obtained using the gene construct illustrated in Figure 24. The construct was ordered from and synthesized by GenScript.

Figure 24: A schematic illustration of the gene construct used to obtain the two different versions of the LHCGR gene: one fused with the native human signal peptide sequence and one fused with the yeast Ste3 signal peptide sequence.


Obtaining and cloning the yLHCGR and the hLHCGR gene

The yeast signal peptide version of the receptor gene, hereafter referred to as “yLHCGR”, was obtained with PCR using the original gene construct (Figure 24) and 5’ phosphorylated primers (lhcgr-rev and lhcgr-fw), as can be seen in Figure 25 A. The yLHCGR construct was then religated and when cut with restriction enzymes NotI and PacI and run on a gel, the obtained band displayed a correct length of approximately 2100 bp (see Figure 25 C). The band was cut out and the yLHCGR gene was then ligated into the shuttle plasmid pSP-GM1 and amplified in E. coli.

The human signal peptide version of the receptor gene, hereafter referred to as “hLHCGR”, was obtained by digesting the original gene construct with NotI and PacI (Figure 25 B). Gel electrophoresis was run and the obtained fragment displayed a correct length of approximately 2100 bp (see Figure 25 C). The band was cut out, ligated into the pSP-GM1 plasmid and amplified in E.coli.

Figure 25: Cloning and isolating the yLHCGR and hLHCGR genes. A) A schematic illustration of how the yLHCGR gene was obtained by PCR using the original gene construct and 5’ phosphorylated primers. The resulting PCR product was religated and then digested with restriction enzymes NotI and PacI and run on a gel in order to verify that the correct product had been obtained. B) The hLHCGR gene was obtained by digesting the original gene construct with NotI and PacI. C) Picture from gel electrophoresis. (L) GeneRuler™ 1 kb DNA Ladder, (1) Digested vector carrying yLHCGR, expected lengths: 2132bp (yLHCGR, has been cut out of the gel) and 2710bp (vector backbone), (2) Digested vector carrying hLHCGR, expected lengths: 2122 bp (hLHCGR, has been cut out of the gel) and 2710 bp (vector backbone), (3) Digested pSP-GM1 plasmid, expected length: 7404bp (has been cut out of the gel)


Saccharomyces cerevisiae strains IMFD-70, IMFD-72, IMFD-73 and IMFD-74 were then successfully transformed with the yLHCGR and hLHCGR expression plasmids respectively. All these strains have been optimized for the coupling of human GPCRs to the pheromone signaling pathway and they differ in their expression of different G alpha proteins. IMFD-70 has the endogenous G alpha protein [1] while the other strains express different versions of yeast/human chimeric G alpha proteins. These chimeric proteins have had the five last amino acid residues of the C terminal end replaced with the corresponding human amino acids [Unpublished data]. IMFD-73 has a chimeric alpha subunit of such type that it should have the best chances of interacting with the LH/CG receptor and was therefore the strain that was mainly used during this project. All strains were kindly provided by the Kondo group at Kobe University, Japan.

Sequencing

In order to confirm that the yLHCGR gene and the hLHCGR gene not only have the correct length but also the correct sequence, the yLHCGR and the hLHCGR plasmid were sent in for sequencing. Results indicated that the sequences were correct for both genes.

Evaluating the functionality of the receptor

The ability of the receptor to detect hCG (i.e to bind the hormone and transmit the signal) when expressed in yeast, was evaluated several times under different conditions. Initial tests were performed by screening for fluorescence after the addition of hCG to the yeast cells. A final test also included screening for indigo production in the completed biosensor system.

Initial tests: Screening for GFP expression

The yeast strains that have been transformed with the gene for the LH/CG receptor have the gene for GFP placed under the control of the pheromone-induced FIG1 promoter. An initial test to see if the LH/CG receptor could detect hCG and activate the pheromone pathway, was therefore done by screening for fluorescence. Below, results from three of these screens are presented.

IMFD-70, IMFD-72, IMFD-73 and IMFD-74, grown at 30°C

One experiment was carried out using all four, IMFD-70, -72, -73 and -74 strains, transformed with yLHCGR, hLHCGR and the empty pSP-GM1 plasmid respectively. Cells were cultivated at 30°C and were treated with lyticase in order to degrade the cell wall. 200 IU of hCG was then added to each strain. Screening for fluorescence was done using a fluorescence microscope. See Figure 3, 4, 5 and 6 for pictures that have been selected as representative of all results obtained during this experiment.

Figure 26: Fluorescence microscopy of the IMFD-70 strain transformed with the yLHCGR plasmid. Cells were grown at 30° C. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 3 hours after the addition of hCG.
Figure 27: Fluorescence microscopy of the IMFD-72 strain transformed with the empty pSP-GM1 plasmid. Cells were grown at 30° C. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 3 hours after the addition of hCG.
Figure 28: Fluorescence microscopy of the IMFD-73 strain transformed with the yLHCGR plasmid. Cells were grown at 30° C. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 2 hours after the addition of hCG.
Figure 29: Fluorescence microscopy of the IMFD-74 strain transformed with the hLHCGR plasmid. Cells were grown at 30° C. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately3 hours after the addition of hCG.

Results obtained for the cells not treated with hCG and cells transformed with the negative control plasmid, indicate that there is relatively high level of background noise of GFP expressions in all IMFD strains. Generally, the fluorescence intensity is approximately equal between samples that have had the hCG hormone added to them and samples that did not have hCG added to them. It was therefore concluded that the LH/CG receptor, when expressed in these yeast strains and tested under the given conditions, is unable to detect hCG or at least that the receptor it is not functioning in a way that produces a sufficiently strong GFP output signal to be distinguished from the background noise.

IMFD-73, grown at room temperature

In a second test, the transformed IMFD-73 strain with either the yLHCGR, hLHCGR or the empty pSP-GM1 plasmid, were cultivated at room temperature. Gron wth at lower temperatures is often used in heterologous protein production since it may improve protein folding. Cells were then treated with cell wall-degrading enzyme lyticase and 200 IU of hCG was added. Results from the fluorescence microscope can be seen in Figure 30, 31 and 32.

Figure 30: Fluorescence microscopy of the IMFD-73 strain transformed with the hLHCGR plasmid. Cells were grown at room temperature. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 2 hours after the addition of hCG.
Figure 31: Fluorescence microscopy of the IMFD-73 strain transformed with the empty pSP-GM1 plasmid. Cells were grown at room temperature. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 2 hours after the addition of hCG.
Figure 32: Fluorescence microscopy of the IMFD-73 strain transformed with the yLHCGR plasmid. Cells were grown at room temperature. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 2 hours after the addition of hCG.

Once again, high levels of background fluorescence were present. Results indicated that also when cells are grown at room temperature, the LH/CG receptor is unable to detect hCG or uncapable to produce an output signal that can be distinguished from the background noise in response to detecting hCG.

IMFD-73-ΔCWP2, grown at 30°C

In a third test, the deletion strain IMFD-73-ΔCWP2 transformed with yLHCGR, hLHCGR and the empty pSP-GM1 plasmid respectively was used. The IMFD-73-ΔCWP strain potentially has a weakened cell wall. Cells were grown at 30°C and were not treated with lyticase before the addition of 200 IU hCG. Fluorescence microscope pictures can be seen in Figure 33, 34 and 35.

Figure 33: Fluorescence microscopy of the IMFD-73 ΔCWP2 strain transformed with the hLHCGR plasmid. Cells were grown at 30° C. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 3 hours after the addition of hCG.
Figure 34: Fluorescence microscopy of the IMFD-73 ΔCWP2 strain transformed with the empty pSP-GM1 plasmid. Cells were grown at 30° C. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 3 hours after the addition of hCG.
Figure 35: Fluorescence microscopy of the IMFD-73 ΔCWP2 strain transformed with the yLHCGR plasmid. Cells were grown at 30° C. GFP fluorescence is shown on the right. The upper panel shows cells that have not been treated with hCG. The lower panel shows cells that have had 200 IU hCG added to the sample. Photos are taken approximately 3 hours after the addition of hCG.

As previously, the yeast transformed with the LH/CG receptor gene and that had the hCG hormone added to them, did not produce an output signal that could be distinguished from the fluorescence intensity of cells that did not have hCG added to them or that did not contain the LH/CG gene.

Final test of the completed system: screening for color change

One last test was performed using the completed biosensor system: IMFD-73-ΔCWP2+pFig-Indigo+hLHCGR or yLHCGR. The cultures were grown over night before adding hCG in the morning. 3 hours later more hCG was added. From the colorometic assay (see "Results: indigo"), it was concluded that it takes approximately 7 hours for the pIndigo cells to produce a visible color change when grown at 30°C. However, 7 hours after adding hCG to the finalized system, no color change could be observed in any of the cultures (see Figure 36 and 37) and it was concluded that the biosensor did not succeed in detecting the hormone.

Figure 36: Test of the finalized system. A) [IMFD-73+hLHCGR+pFig-Indigo]. hCG was added to the sample to the left, while sample to the left were left untreated. B) [IMFD-73+hLHCGR+pIYCO4]. hCG was added to the sample to the left, while sample to the left were left untreated.
Figure 37: Test of the finalized system. A) [IMFD-73+yLHCGR+pFig-Indigo]. hCG was added to the sample to the left, while sample to the left was untreated. B) [IMFD-73+yLHCGR+pIYCO4]. hCG was added to the sample to the left, while sample to the left was untreated.

Discussion of results and future perspectives

Expression of the gene for the LH/CG receptor in yeast

When analyzing our results, we have identified several possible reasons that may have lead to the biosensor being unable to detect the hCG hormone. Below, we discuss these and also present some ideas on how to take our project further and improve our study.

1. The hCG hormone could not pass the yeast cell wall

We were aware of the risk that the hCG hormone, due to its size and due to the limited permeability of the yeast cell wall, would not be able to pass the cell wall and therefore not reach the receptor. This is the reason that the ΔCWP2 strain was created and that the initial fluorescence screenings were done using cells that had been treated with cell wall-degrading enzymes. Despite these efforts, there is a small risk that the hCG hormone never reached the receptor, which would then explain the lack of a successful detection. However, we deem this to be relatively unlikely. At least the lyticase treatment should have allowed some hCG to reach the receptor.

If, however, further investigations would show that the cell wall permeability of the Δcwp2 strain is still too limited for hCG to pass, one could also try to delete the gene CWP1 in order to increase the permeability additionally [2]. But there are also several other possibilities to weaken the cell wall, for example the deletion of genes whose corresponding proteins contribute to the assembly of the cell wall or knock-out of other cell wall components.

2.The receptor only mediates weak signals

The receptor might be able to bind hCG but, due to inefficient interaction between the receptor and the yeast G alpha, only partially induce the activation of the pheromone pathway. This is a common problem when expressing human GPCRs in yeast and that is the reason why strains with chimeric G alpha proteins have been used when expressing the LH/CG receptor. Since these chimeric G alpha proteins have had the last 5 C terminal amino acid residues of the yeast G alpha protein replaced by the corresponding human amino acids, they should enable the human receptor to couple with the G alpha subunit while still preserving the G alpha ability to interact with G betagamma complex. Still, it might be so that the LH/CG receptor interacts inefficiently with the yeast G protein and thus only produces a very weak output signal. This may then be the reason why our biosensor was unable to produce a noticeable signal in response to detecting hCG.

If the LH/CG receptor only produces a weak output signal, the background noise of GFP expression when examining our cells in the fluorescence microscope would have made it difficult to identify cells that actually displayed signal-induced expression of GFP. To improve analysis, a next step could therefore be to use flow cytometry. This method enables comparative quantification of the GFP signaling in whole cell populations [3]and may then enable even weak signal-induced GFP expression to be distinguished from the background noise.

Another possible step to solve the potential problem of a weak signal would be to amplify the signal. A method designed to this was developed by Fukada et al in 2011. Their strategy involves invoking positive feedback in response to activation of the signaling pathway. This is done by placing both the gene for the yeast G beta subunit and the reporter gene under the control of the FIG1 promoter. Thus, when a ligand binds to the receptor, the Gbeta is overexpressed and can compete with the endogenous Gbeta for bindinf the G alpha subunit. This competition would allow G betagamma complexes to be released and to activate the signaling pathway once again thereby amplifying the weak signal and increasing the expression of the reporter gene [4].

3. The receptor is not functional when expressed in yeast

There is always a risk when expressing a protein in another host that the protein will not be functional. This might be due to an incorrect fold or that the protein did not get the proper post-translational modifications. The LH/CG receptor, when expressed in yeast, might have lost its correct conformation, been unable to integrate properly into the yeast cell membrane and therefore also be unable to bind the hCG hormone. Mammalian proteins will be glycosylated in a significantly different manner when expressed in yeast. This might be a potential problem for the LH/CG receptor, which has a number of glycosylated sites. According to literature, these glycosylations are not important for ligand binding and signal transduction, but they might be important for proper integration of the receptor into the membrane[5]. This may then explain why the receptor is non-functional in yeast.

As a further step, we would like to determine the location of the LH/CG receptor when expressed in yeast. This could be done by, for example, placing a fluorescent tag on the receptor and would enable us to see whether or not it has been integrated in the yeast membrane or if it has been retained in the cytosol.

If it turns out that LH/CG successfully has been integrated in the membrane but that the receptor is unable to function properly, we would also like to try to reengineer the receptor. In previous studies deletion of foremost intracellular loops has proven to increase the stability and activity of some GPCRs when expressed in yeast[6]. This might be something that could be applied to the LH/CG receptor as well since it does have several large intracellular loops.

References

[1] Togawa F, Ishii J,Ishikura A, Tanaka T, Ogino C, Kondo A. Importance of asparagine residues at positions 13 and 26 on the amino-terminal domain of human somatostatin receptor subtype-5 in signalling. The Journal of Biochemistry. 2010;147(6):867-873.


[2] Zhang M, Liang Y, Zhang X, Xu Y, Dai H, Xiao W. Deletion of yeast CWP genes enhances cell permeability to genotoxic agents. Toxicological sciences: an official journal of the Society

of Toxicology. 2008;130(1):68-76.

[3] Ishii J, Moriguchi M, Hara KY, Shibasaki S, Fukuda H, Kondo A. Improved identification of agonist-mediated Gαi-specific human G-protein-coupled receptor signaling in yeast cells by flow cytometry. Analytical Biochemistry. 2012;426(2):129-133.
[4] Fukuda N, Ishii J, Kaishima M, Kondo A. Amplification of agonist stimulation of human G-protein-coupled receptor signaling in yeast. Analytical Biochemistry. 2011;417(2):182-187.
[5] Petäja-Repo UE, Merz WE, Rajaniemi HJ. Significance of the carbohydrate moiety of the rat ovarian luteinizing hormone/chorionic-gonadotropin receptor for ligand-binding specificity and signal transduction. The Biochemical Journal. 1993;292(3):839-844.
[6] Ladds G, Goddard A, Davey J. Functional analysis of heterologous GPCR signaling pathways

in yeast. Trends in Biotechnology. 2005;23(7):367-373












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