Difference between revisions of "Part:BBa K2213006"

Revision as of 02:17, 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 tag was combined with the low strength Anderson promoter (https://parts.igem.org/Part:BBa_J23105) and mCherry. 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.

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 made by Manchester 2017

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.
The improved part has been assembled under different strength promoters to find the optimal level of induction for localisation in a EUT microcompartment constructed from 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).

• 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 data on this can be seen in the section below.

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 when 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