Difference between revisions of "Part:BBa K2213008"

Revision as of 00:39, 2 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.

Figure1. circuit diagram of BBa_K2213006.
The PduD(1:20) tag is one of many tags that has been proved to localise inside a Pdu microcompartment. This was proved by the iGEM Dundee 2011 team http://2011.igem.org/Team:Dundee. To develop on this, Manchester2017 Characterised the localisation of this tag within a EUT microcompartment consisting of BBa_K2213000, BBa_K2213001 and BBa_K2213002. (https://parts.igem.org/Part:BBa_K2213000 https://parts.igem.org/Part:BBa_K2213001 https://parts.igem.org/Part:BBa_K2213002)

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 high strength Anderson promoter (here)
• low strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213006)
• medium strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213007)

Characterisation

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





Figure 4. Fluorescence microscopy images of High strength Anderson promoter-PduD construct associated mCherry when expressed alone and with Eut subunits.

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