Team:Manchester/Results4

Three different strength Anderson promoters were each combined with the PduD localisation tag to enable variable synthesis of PduD and allow us to optimise localisation.

We aimed to confirm the function of the different PduD-Anderson promoter constructs by expressing them with mCherry attached so that they could be visualised via fluorescent microscopy. We visualised the different strength tag-promoter constructs, using mCherry fluorescence to check the expression level of each construct.

Biobricks used are as follows:
BBa_K2213006: LowPromoter_PduD(1-20)_mCherry (Low)
BBa_K2213007: MediumPromoter_PduD(1-20)_mCherry (Medium)
BBa_K2213008: HighPromoter_PduD(1-20)_mCherry (High)

Figure 1. Optical Density (600nm) for Low (BBa_K2213006), Medium (BBa_K2213007) and High (BBa_K2213008) strength Anderson promoter constructs with RFU values after 30 hours. Dotted lines represent 95% confidence intervals as calculated from 12 replicates.

As expected, the level of fluorescence increases with each strength promoter. The difference in fluorescence was most pronounced between low promoter and the other two. It is important to note that the expression levels of the medium and high strength promoters are very similar. This result was unexpected and should be taken into consideration when choosing a suitable promoter strength.

The different tag expression levels under each promoter was further investigated via fluorescence microscopy.

Figure 2. Fluorescence microscopy images of Low, Medium and High strength Anderson promoter-PduD construct associated mCherry (OD600: 0.2) expressed in the absence of Eut.

As shown, there is a gradient of fluorescence, substantiating the previous results. These results demonstrate proper functioning of the low promoter and highlight the irregular expression levels between the medium and high promoters, suggesting one or both are not functioning as intended.