Part:BBa_K638403:Experience

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Applications of BBa_K638403

This part was intended to demonstrate successful export of a reflectin fusion to the periplasm by the TAT pathway in E coli, using our modified torA tag, which could be demonstrated by seeing GFP localised to the periplasm of an E coli cell.

We built this construct in a pSB3K3 backbone, transformed into DH10B cells. We [http://2011.igem.org/Team:Cambridge/Protocols/Preparing_Slides_With_Agarose_Supplement_To_Form_Microcolonies grew these cells on agar pads] with 1mM arabinose and imaged them with a confocal microscope (with the help of Paul Grant). We saw diffuse fluorescence throughout the cell, showing no evidence of successful export to the periplasm.

Further work: Most E coli strains, including DH10B, exhibit an 'all-or-nothing response' to arabinose as described [http://www.ncbi.nlm.nih.gov/pubmed/11739756 here], and we induced our cells at a high level. Export pathways can easily become saturated, and our findings do not rule out the possibility that both cytoplasm and periplasm were saturated with reflectin-GFP. We would like to repeat this work with a strain that exhibits a titratable response to arabinose, at a lower level of induction (1-5 μM arabinose).

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