Difference between revisions of "Part:BBa K2213013"

Revision as of 03:51, 2 November 2017

araB_eutLK_LowPromoter_PduD(1-20)_mCherry_cgPPK2
araB_eutLK_LowPromoter_PduD(1-20)_mCherry_cgPPK2 is a composite of https://parts.igem.org/Part:BBa_K2213002 and https://parts.igem.org/Part:BBa_K2213009.

Figure 1: Schematic overview of araB_eutLK_LowPromoter_PduD(1-20)_mCherry_cgPPK2 (BBa_K2213013)

Characterisation

This part was con-transformed with EutSMN (https://parts.igem.org/Part:BBa_K2213012) to form EutSMNLK+Low_PduD+PPK which was characterized as follows.

A 24 hour induction was DAPI stained to determine microcompartment formation, tag localisation and PPK activity.


Figure 2: Fluorescence microscopy images of promoter-PduD associated mCherry and DAPI stained polyphosphate.

Figure 3. Visible light, mCherry and DAPI signals from DAPI stained E. coli, lacking PPK.

DAPI staining and mCherry fluorescence within E.coli appear to colocalised, and locally enriched to foci at particular sub-cellular sites (Figure 2). This heterogeneous distribution of DAPI is induced by the presence of promoter-PduD-mCherry-PPK, with controls lacking the PPK element showing largely ubiquitous DAPI-staining throughout the cell (Figure 3). We conclude these results to indicate the presence of polyphosphate, and prove the activity of the PPK along with its successful localisation into the Eut microcompartment. These findings help establish a proof-of-concept functionality of the system and demonstrate successful Eut subunit expression, successful tag localisation and successful PPK activity.

Sequence and Features