Team:Manchester/Demonstrate
Proof of Concept
Microscopy
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.
From literature we learned that polyphosphate chains can be stained using DAPI, which can then be visualised using a separate excitation wavelength from mCherry. 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 PPK
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) and no expression of Eut microcompartments. Our choice of control cells also allowed us to confirm that we could visualise both signals from the same cells.
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 with EutSMN:
As shown, there is a heterogeneous distribution of fluorescence within the cells for both signals and they are approximately in the same areas. This heterogeneous distribution of DAPI indicates the presence of polyphosphate and proves the activity of our PPK along with its successful localization into our bacterial microcompartment. The localisation can be determined using the physical location of both fluorescence signals within the cell.
