Team:Manchester/Results3

We aimed to confirm that our Eut microcompartments and PduD localisation tag were compatible. We visualised the tag-promoter constructs via fluorescence microscopy using mCherry to check the distribution of the tag throughout the cell and investigated whether the tag is localised in the presence of Eut subunits.

(As medium promoter and high promoter had similar levels of expression, we decided to just use low and high in combination with Eut subunits for characterisation).

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)
BBa_K2213000: LacUV5_EutS (EutS)
BBa_K2213012: LacUV5_EutS_TetR_EutMN (EutSMN)
BBa_K2213013: araB_eutLK_LowPromoter_PduD(1-20)_mCherry_cgPPK2 (EutLK-Low-PduD-mCherry-PPK)

Figure 1. Fluorescence microscopy images of Low and High strength Anderson promoter-PduD construct associated mCherry expressed alone and co-expressed with Eut subunits.

Levels of fluorescence from Low+EutS were too low to properly visualize. High+EutS showed slightly heterogeneous distribution of fluorescence, the fluorescence was slightly granular with some brighter areas and some darker areas but no well-defined localisation.

Low+EutSMN images were very dim but visualization was possible. The fluorescence was granular but mainly homogeneous throughout the cell. High+EutSMN showed quite well defined localisation in a number of cells.

EutSMNLK was visualized by combining EutLK-Low-PduD-mCherry-PPK and EutSMN. Fluorescence from Low+EutSMNLK showed quite well-defined localisation with a number of cells showing relatively round accumulations of fluorescent tag, suggesting proper BMC formation.

(It is important to note that the majority of the accumulations seen occur at or near the end of the cells. This could be indicative of protein aggregates and not proper BMC formation (Bednarska et al., 2013)).

An added benefit of our SMNLK+PPK construct is that it would allow us to determine whether it actually worked via DAPI staining. The polyphosphate chains produced by PPK can be DAPI stained and visualised by setting the excitation filter at 370nm and emission filter at 526nm, and as such both mCherry and DAPI could be visualised in the same cells. This would allow us to see any co-localisation of the two signals, demonstrating successful Eut subunit expression, successful tag localisation and successful PPK activity.

By modifying the protocol found here, we were able to DAPI stain our cells (our modified protocol can be found here). A control DAPI stain was performed along side Medium promoter tag expression to inspect the DAPI distribution in the absence of polyphosphate and whether the staining procedure interfered with mCherry distribution.

Figure 2. Visible light, mCherry and DAPI signals from DAPI stained E. coli expressing Medium strength Anderson promoter-PduD construct.

As shown, the distribution of both mCherry and DAPI were homogeneous with no obvious clumping or accumulation. This suggested that if our construct was working properly, we would see an accumulation of fluorescent signal for both mCherry and DAPI in the same place within the cell.

So in line with this, we DAPI stained a 24 hour induction of Low+EutSMNLK+PPK:

Figure 3. Fluorescence microscopy images of promoter-PduD associated mCherry and DAPI stained polyphosphate.

This heterogeneous distribution of DAPI indicates successful dying of polyphosphate and confirms the activity of our PPK along with its successful localisation into our bacterial microcompartment. These findings demonstrate the proof-of-concept functionality of our Phosphostore system.

More detailed information can be found in our Demonstration page here.