Team:Chalmers-Gothenburg/Results

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. For instance, blue bubbles could be seen in one medium and other media turned into different shades of brown. However, we did not manage to confirm the presence of bio-indigo and to exactly determine 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 of the results can be found in the sections below.

Survival of yeast in urine

Figure 1: Growth curve for cells in Urine and YPD medium. One can see that the OD remains the same in the urine i.e. that 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. Pre-cultures of a normal lab yeast strain were prepared in 5ml YPD and let 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 37°C. OD600 measurements were taken at different points of time during the day, and the values can be seen in Figure 1. The cells in urine did not grow; the OD remained the same during the whole day, while the cells in YPD grew very well.

Figure 2: Spot test analysis of cells grown in YPD and urine for 4h. The cells survive in the urine and grow normal after plating on a YPD plate.

In order to test if the cells still are alive in the urine medium, 300 µl of each culture (after 4h) were taken and OD 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 4h in urine and grew normal after being spotted on a YPD plate. Summarizing, Yeast cells are unable to bud/divide in the urine medium but survive under these conditions.

Deletion of CWP2 gene

Gene Deletion

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 higher chances of our ligand to pass the cell wall and to bind to the membrane-bound receptor. The gene deletion was performed according to the bipartite method. The results from the first PCR reactions, where 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 vectors, are amplified by PCR (PCR reaction 1 and 2). In addition, 500 bp of the up- and downstream sequence of the CWP2 gene are amplified from genomic DNA using primers with 5' extensions that are complement to the loxP sites as indicated with lines in the schematic illustration above. B) The results of the four PCR reactions on gel. The expected sizes of the kanMX and CWP2 fragments are 1000 bp and 500 bp respectively which correspond to the sizes observed on the gel.

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

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

Colony PCR

Yeast was transformed with 150 ng of each fragment and then let grown on G418 medium. Colony PCR was performed with a forward primer outside of the inserted region and reverse primers within the CWP2 gene or KanMx cassette respectively and the deletion could be confirmed (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 has been used in the construction of the bipartite fragment. Two reverse primers were designed to hybridize within the CWP2 gene and within the kanMX cassette respectively. The primers are indicated with arrows. B) The results of the PCR reactions on gel. In every reaction, the same cwp2-fw primer was used 1: The Δcwp2 deletion strain (A, bipartite), PCR with the kanMX-rev primer. 2: The Δcwp2 deletion strain (B, from deletion library) with the kanMX-rev primer. 3: The Δcwp2 deletion strain (A, bipartite), PCR with the cwp2-rev primer. 4: The Δcwp2 deletion strain (B, from deletion library, PCR) with the cwp2-rev primer. 5. A wild type yeast strain, PCR 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) but no bands should be observed when PCR is performed with the cwp2-rev primer (3, 4). On the contrary, a clear band should be observed when running PCR with the wild type strain and the cwp2-rev primer. This corresponds to the bands displayed on the gel. This indicates that the deletion was successful.

Lyticase assay

A lyticase assay was performed primarily in order to check the activity of the 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 point of times. 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 are intact between 30 and 60 minutes (except for one outlier at 50 min) which means that the cell wall degrades quicker in the [IMFD-73 Δcwp2::kanMX] strain. This leads us to the conclusion that the cell wall is somewhat weakened in the strains with the deleted cell wall mannoprotein 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.

Expression of human LH/CG receptor

Introduction of indigo synthesizing genes

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