Team:Chalmers-Gothenburg/Results

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.  

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.  

=='''Survival of yeast in urine'''==  

=='''Survival of yeast in urine'''==  

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 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. The growth curve 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 were growing very well.

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 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. The growth curve 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 were growing very well.

[[File:Spottest-urine2.jpg|thumb|250px|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.]]

[[File:Spottest-urine2.jpg|thumb|250px|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.]]

=='''Deletion of ''CWP2'' gene'''==

=='''Deletion of ''CWP2'' gene'''==

'''Gene Deletion'''

'''Gene Deletion'''

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.  

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.  

[[File:Resultpcr1.jpg|thumb|600px|center|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.]]

[[File:Resultpcr1.jpg|thumb|600px|center|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.]]

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 Δ''cwp2''::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 Δ''cwp2''::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''.

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 Δ''cwp2''::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 Δ''cwp2''::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''.

[[File:Lyticaseassay1.jpg|thumb|500px|center|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.]]

[[File:Lyticaseassay1.jpg|thumb|500px|center|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.]]

=='''Expression of human LH/CG receptor'''==

=='''Expression of human LH/CG receptor'''==

=='''Introduction of indigo synthesizing genes'''==

=='''Introduction of indigo synthesizing genes'''==