Designed by: Philipp Lorenz   Group: iGEM14_Oxford   (2014-09-29)

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Applications of BBa_K1446002

We have used this part in order to test whether the targeting mechanism of the N-terminal tag works. This has been achieved by coexpressing this microtagged sfGFP (in the pRSFDuet expression vector) along with the pdu-microcompartment submitted by the Dundee team in 2009 ( BBa_K562009 ) in the pUNI-vector (Amp-resistance, tat-promoter). We managed to get fluorescence images indicating that the targeting mechanism does indeed work in some of the cells as shown in the images below:

The image on the left shows the high fluorescent density spots, whereas the image on the right is from a control sample with no microcompartment-expressing vector. Both images are taken from the same sample with the biobrick being expressed in the pRSFDuet vector, which has kanamycin resistance and a promoter under lac-control. The liquid cultures were grown overnight (16 hours). In the morning, 1 in 200 dilutions were made (with fresh antibiotics) with IPTG concentrations of 0, 5, 10 and 100 & micromolar. The images shown here are from the cultures with 100 micromolar IPTG after growing them for 2 hours (OD of 0.568). Exposure time is 0.3 seconds.

As can be seen for some of the cells in the left image, there is no perfect and uniform distribution of sfGFP, but clearly identifiable spots of higher fluorescence densities which suggests that the targeting mechanism of the N-terminal tag has worked successfully. For the image analysis we used the ImageJ software, splitting the channels into red, green and blue and selected images from the green channel.

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