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, https://parts.igem.org/Part:BBa_K2213006 and https://parts.igem.org/Part:BBa_K2213003.

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

Characterisation

This part was expressed alongside 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.

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 the PPK along with its successful localization into the Eut microcompartment. The localisation can be determined using the physical location of both fluorescence signals within the cell. 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1300
  Illegal NheI site found at 2701
  Illegal NheI site found at 2724
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1239
  Illegal BamHI site found at 2164
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2839
  Illegal SapI site found at 1056
  Illegal SapI.rc site found at 4280