Difference between revisions of "Part:BBa K833003"

Latest revision as of 04:26, 4 October 2012

mutated prfA-UTR thermosensor from Listeria monocytogenes

This part was derived from part BBa_K619889 via TAQ random mutagenesis and screening for thermosensors that responded to a 35 to 37'C shift. In contrast to the our wild type thermosensor (part BBa_K619889) which activates translation from a 30 to 37 degree shift, this thermosensor activates transcription from a 35 to 37 degree shift (a more concise dynamic range). This mutation was isolated by randomly mutagenizing part BBa_K619889 and monitoring a lacZ output. The thermosensor has a shine delgarno and initiating ATG that are barried within a stem-loop structure at 30'C, but are accessible at higher temperatures as the mRNA structure is disrupted. This thermosensor is part of a library of thermosensors from this screen (see parts (Bba_K833000 to Bba_K833010 and also BBa_K619890,BBa_K61989 -619894 from our 2011 submission). mutated prfA thermosensor from listeria D: This is the prfA-UTR from Listeria monocytogenes which has been randomly mutated to produce a thermosensor this a more discrete dynamic range.

To use: fuse a reporter (such as LacZ, GFP or RFP) to the initiating ATG in frame

Plate assay: In contrast to the our wild type thermosensor (part BBa_K619889) which activates translation from a 30 to 37 degree shift, this thermosensor activates transcription from a 35 to 37 degree shift. This phenotype has been confirmed 3 times on X-gal plates using the LacZ reporter and also in liquid B-gal assays. There is essentially no LacZ at 30'C in contrast to our original part BBA_K619889. .

Sequence and Features