Team:Manchester/Demonstrate

We aimed to demonstrate the functionality of our Eut-PPK-tag system by visual means. By using a DAPI stain, mCherry and fluorescence microscopy, we could distinguish tag expression, tag localisation within the Eut microcompartment and polyphosphate production all from the same microscope slides.

We learned that polyphosphate chains can be stained using DAPI from this journal article in Nature, which can then be visualised by setting the excitation filter at 370nm and emission filter at 526nm. This meant that using two different filters would allow both the DAPI signal and the mCherry signal from cells to be pictured.

In theory these two signals would overlap as the mCherry tagged PPK would produce a polyphosphate chain, which would be stained with DAPI, both of which would be found localised within the Eut microcompartment.

Control experiment with no expression of Eut Microcompartment

To test DAPI’s spatial distribution within cells after staining, we performed a control stain in E. coli which only express the medium promoter tag (BBa_K2213007) with no expression of any Eut microcompartment subunits or PPK. Our choice of control cells also allowed us to confirm that we could visualise both signals from the same cells.

Figure 1. Visible light, mCherry and DAPI signals from DAPI stained E. coli expressing Medium strength Anderson promoter-PduD construct.

As shown, the distribution of DAPI in the absence of polyphosphate was roughly homogeneous with no obvious clumping or accumulation. The same homogeneous distribution of mCherry was also observed, demonstrating that the DAPI stain does not interfere with the distribution of the tag. This suggested that, if working properly, images of our construct would display clear accumulation of both mCherry and DAPI signals in the same location within the cell.

DAPI-stained polyphosphate localises around mCherry tagged PPK targeted to BMC

So in line with this, we DAPI stained a 24h induction of EutLK-Low-PduD-mCherry-PPK (BBa_K2213013) with EutSMN (BBa_K2213012):

Figure 2. 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 help establish a proof-of-concept functionality of our Phosphostore system and demonstrate successful Eut subunit expression, successful tag localisation and successful PPK activity.