Difference between revisions of "Part:BBa K2213006"
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| − | <b>Figure 3.</b> | + | <b>Figure 3.</b> Optical Density (600nm) for Low, Medium and High strength Anderson promoter constructs with RFU values after 30 hours.<br> |
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The expression levels compared to medium and high are as shown.<br> | The expression levels compared to medium and high are as shown.<br> | ||
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| − | <b>Figure 4.</b> | + | <b>Figure 4.</b> Fluorescence microscopy images of Low strength Anderson promoter-PduD construct associated mCherry expressed alone and with Eut subunits.<br> |
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When expressed alone the distribution of the mCherry signal was homogeneous and fluorescence level was very low making visualisation difficult. In the presence of EutSMNLK the mCherry signal was much brighter and clumped together, indicating localisation to the BMC. | When expressed alone the distribution of the mCherry signal was homogeneous and fluorescence level was very low making visualisation difficult. In the presence of EutSMNLK the mCherry signal was much brighter and clumped together, indicating localisation to the BMC. | ||
Revision as of 01:49, 1 November 2017
LowPromoter_PduD(1-20)_mCherry
This part is an improved version of https://parts.igem.org/Part:BBa_K562001, submitted by Dundee 2011.
The PduD(1:20) tag is one of many tags that has been proved to localise inside a Pdu microcompartment. This has been proved by the iGEM Dundee 2011 team http://2011.igem.org/Team:Dundee. This part has been tagged with the fluorescent protein, mCherry, to facilitate its characterisation, as can be seen in the figure below:
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.
- A low strength Anderson promoter (here)
- medium strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213007)
- high strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213008)
Characterisation
The PduD tag was combined with the low strength Anderson promoter (https://parts.igem.org/Part:BBa_J23105) and mCherry.
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 medium and high promoters.
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 medium and high are as shown.
Figure 4. Fluorescence microscopy images of Low strength Anderson promoter-PduD construct associated mCherry expressed alone and with Eut subunits.
When expressed alone the distribution of the mCherry signal was homogeneous and fluorescence level was very low making visualisation difficult. In the presence of EutSMNLK the mCherry signal was much brighter and clumped together, indicating localisation to the BMC.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 8
Illegal NheI site found at 31 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]