Difference between revisions of "Part:BBa K638403:Experience"

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===Applications of BBa_K638403===
 
===Applications of BBa_K638403===
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It was hoped that this part would demonstrate successful export of a reflectin fusion to the periplasm by the TAT pathway in E coli, using our modified [[Part:BBa_K638402 | torA tag]], which would be demonstrated by seeing GFP localised to the periplasm of an E coli cell. We built this construct in a [[Part:pSB3K3 | pSB3K3 backbone]] and transformed into DH10B cells. Images of these cells taken on a confocal microscope (with the help of Paul Grant) showed diffuse fluorescence throughout the cell, suggesting that export was '''not successful''' -- Cambridge iGEM 2011
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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 [[Part:BBa_K638402 | torA tag]], which could be demonstrated by seeing GFP localised to the periplasm of an E coli cell.
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We built this construct in a [[Part:pSB3K3 | pSB3K3 backbone]], transformed into DH10B cells. We [[Team: 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.
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'''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]. We would like to try this construct in a strain that exhibits a titratable response to arabinose, at a lower level of induction (1-5 μM arabinose).
  
 
===User Reviews===
 
===User Reviews===
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Revision as of 14:23, 20 September 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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 Team: 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]. We would like to try this construct in a strain that exhibits a titratable response to arabinose, at a lower level of induction (1-5 μM arabinose).

User Reviews

UNIQ060b26c7729417a3-partinfo-00000000-QINU UNIQ060b26c7729417a3-partinfo-00000001-QINU