Testing Biobrick E2050 Part 2 - FACS Analysis of mOrange recombinant pRS415

Aim

The aim of this experiment was to test if mOrange inserted into pRS415, (mOrpRS415) in place of green fluorescent protein, (GFP) will fluoresce when galactose is added.

Hypothesis

Since GFP has been detected when pRS415 is induced with galactose, in-frame insertion of mOrange DNA sequence in place of GFP in pRS415 should produce orange fluorescence when galactose is added. This will allow confirmation of whether the Bio-brick E2050 will work.

Protocol

Starter cultures of BY4741, BY4741 pRS415 and BY4741 mOrpRS415 were incubated overnight and the OD600 was measured. These were then used to set-up overnight experimental cultures to have an OD600=0.6 for the following day. The following were set-up as shown by (Table 1).

The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the FACs analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively as can be seen from the following data obtained from the BD Fluoresence Spectrum Viewer.

Results

From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.

To check the background fluorescence of BY4741, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of pRS415 BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.

The results are shown in Fig.1(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (pRS415 BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid pRS415 or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.

An analysis for mOrpRS415 sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.1(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.2(ii), which suggests that the fluorescence detected is GFP, which was not expected.

In a previous experiment, PCR screening was used to establish that the transformed colonies were positive for mOrpRS415, which makes the chances of a BY4741 transformed with uncut and non-homologously recombined pRS415 with mOrange insert unlikely.

Despite this, the observation of a small peak for both FITC and PE-A with the features as discussed above suggests that there are some yeast cells within the mOrpRS415 BY4741 samples expressing GFP rather than mOrange. Which implies that the homologous recombination was not 100% efficient.

Conversely, 83.4% of the analysed cell population did not show any fluorescence, which suggests that either mOrange was not successfully homologously recombined or that it could not be expressed by BY4741. If uncut pRS415 had been transformed in place of mOrpRS415 it is likely that a greater GFP fluorescence would have been detected, which was not the case. The cut pRS415 does not have any complementary ends that could re-ligate. Therefore it is unlikely that these are transformed and selected.

Ideally, a positive control for detecting mOrange would allow verification of the specificity of PE-A detection of mOrange and a DNA sequence of transformant of mOrpRS415 would confirm whether or not the yeast carried the appropriate recombined plasmids.

Conclusions

From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in mOrpRS415 BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in pRS415 was not 100% efficient and further experimentation is required to confirm this result.