Translational_Unit

Part:BBa_K2213008

Designed by: Adam Hannaford   Group: iGEM17_Manchester   (2017-10-08)
Revision as of 02:06, 1 November 2017 by AmberJam (Talk | contribs)


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.



Characterisation


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

PromoterComparison800p.png

Figure 2. Fluorescence microscopy images of Low, Medium and High strength Anderson promoter-PduD construct associated mCherry (OD600: 0.2) expressed in the absence of Eut.

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



TagExpression500p.jpg
Figure 3. Optical Density (600nm) for Low, Medium and High strength Anderson promoter constructs with RFU values after 30 hours.

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

Figure 4. Optical Density (600nm) for Low, Medium and High strength Anderson promoter constructs with RFU values after 30 hours.

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.


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]


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Categories
Parameters
None