Difference between revisions of "Part:BBa K2213008"
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https://static.igem.org/mediawiki/parts/2/24/Manchesterigem17-high-tag1.png
 
https://static.igem.org/mediawiki/parts/2/24/Manchesterigem17-high-tag1.png
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<br>
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<b>Figure1.</b> circuit diagram of BBa_K2213006.
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<br>
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===Improvements===
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<br>
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This part is an improved version of https://parts.igem.org/Part:BBa_K562001, originally submitted by team Dundee 2011. The original part contained an illegal XbaI site which has been removed to make it biobrick compatible.
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<br>
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This part has also been expressed under different strength promoters, by Manchester2017, to find the optimal level of induction.
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<br>
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<ul>
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<li>A high strength Anderson promoter (here)</li>
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<li>low strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213006)</li>
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<li>medium strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213007)</li>
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</ul>
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<br>
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<br>
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Revision as of 02:03, 1 November 2017


HighPromoter_PduD(1-20)_mCherry

This part is an improved version of https://parts.igem.org/Part:BBa_K562001, submitted by Dundee 2011.

Manchesterigem17-high-tag1.png
Figure1. circuit diagram of BBa_K2213006.

Improvements


This part is an improved version of https://parts.igem.org/Part:BBa_K562001, originally submitted by team Dundee 2011. The original part contained an illegal XbaI site which has been removed to make it biobrick compatible.
This part has also been expressed under different strength promoters, by Manchester2017, to find the optimal level of induction.




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 8
    Illegal NheI site found at 31
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Function:
This part is an improved version of https://parts.igem.org/Part:BBa_K562001, originally submitted by team Dundee 2011. The original part contained an illegal XbaI site which has been removed to make it biobrick compatible. This part has been expressed under different strength promoters. A high strength Anderson promoter here, low strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213006), and a medium strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213007).

The PduD tag was combined with the high strength Anderson promoter (https://parts.igem.org/Part:BBa_J23104) and mCherry.

PromoterComparison800p.png

A gradient of fluorescence is evident when compared to low and medium promoters.



TagExpression500p.jpg
The expression levels compared to low and medium are as shown. Fluorescence levels are similar to the medium promoter, this result was unexpected and should be taken into consideration when choosing either the medium or high promoter.



HighPromoterComparison800p.png

When expressed alone the distribution of the mCherry signal was homogeneous and fluorescence level was relatively high. In the presence of EutS the mCherry signal was less homogeneous but lacked obvious localisation, suggesting EutS isn’t enough to form proper BMCs. In the presence of EutSMN the mCherry signal clumped together, indicating localisation to the BMC.