Part:BBa_K2213013
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 be co-localised, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1300
Illegal NheI site found at 2701
Illegal NheI site found at 2724 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1239
Illegal BamHI site found at 2164 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1074
Illegal AgeI site found at 1775
Illegal AgeI site found at 1952
Illegal AgeI site found at 2252
Illegal AgeI site found at 2315
Illegal AgeI site found at 2362
Illegal AgeI site found at 3601 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2839
Illegal SapI site found at 1056
Illegal SapI.rc site found at 4280
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