Difference between revisions of "Part:BBa K2213013"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2213013 short</partinfo>
 
<partinfo>BBa_K2213013 short</partinfo>
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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.
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<br>
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https://static.igem.org/mediawiki/2017/f/fa/LK-Low-tag-mcherry-PPK.png
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<br>
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<b>Figure 1:</b> Schematic overview of araB_eutLK_LowPromoter_PduD(1-20)_mCherry_cgPPK2 (BBa_K2213013)
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===Characterisation===
 
===Characterisation===
  
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This part was expressed alongside EutSMN (https://parts.igem.org/Part:BBa_K2213012) to form EutSMNLK+Low_PduD+PPK which was characterized as follows.<br>
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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.<br>
 
<br>
 
<br>
 
A 24 hour induction was DAPI stained to determine microcompartment formation, tag localisation and PPK activity.<br>
 
A 24 hour induction was DAPI stained to determine microcompartment formation, tag localisation and PPK activity.<br>
 
<br>
 
<br>
 
https://static.igem.org/mediawiki/2017/4/4f/LowSMNLKDAPIcells.png<br>
 
https://static.igem.org/mediawiki/2017/4/4f/LowSMNLKDAPIcells.png<br>
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<b>Figure 1.</b> Fluorescence microscopy images of promoter-PduD associated mCherry and DAPI stained polyphosphate.<br>
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<b>Figure 2:</b> Fluorescence microscopy images of promoter-PduD associated mCherry and DAPI stained polyphosphate.<br>
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<br>
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<br>
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https://static.igem.org/mediawiki/2017/0/05/Manchesterjessdapi.png<br>
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<b>Figure 3.</b> Visible light, mCherry and DAPI signals from DAPI stained E. coli, lacking PPK.<br>
 
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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.<br>
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DAPI staining and mCherry fluorescence within <i>E.coli</i> 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.<br>
 
<br>
 
<br>
  

Latest revision as of 03:58, 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.
LK-Low-tag-mcherry-PPK.png
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.

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


Manchesterjessdapi.png
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


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