Part:BBa_K611018
Mutant Regulatory protein/promoter characterization plasmid
This part can be used to characterize promoter/repressor and promoter/activator pairs by swapping in the desired parts on either end.
To characterize promoter mutants, ligate the corresponding wild-type repressor or activator on the 3' end by cutting the plasmid with SpeI and PstI and the regulatory protein with XbaI and PstI in preparation for ligration. To add a mutant promoter to the 5' end, cut the construct at EcoRI and XbaI and the promoter at EcoRI and SpeI.
Repressor/activator mutants can also be characterized by simply putting a wildtype promoter on the 5' end and adding the mutant repressor or activator on the 3' end.
Expression is reported via GFP fluorescence. The amount of repressor/activator can be controlled using the arabinose inducible pBAD promoter, with increased levels of arabinose induction leading to higher repressor/activator concentration and a corresponding change in promoter activity level.
Transcriptional termination takes place within the psb1C3 plasmid which means it is not necessary to add a terminator once the wild-type and mutant sequences have been ligated to either end.
This part was used to build parts like BBa_K611016, which allowed the characterization of the LacI promoter (BBa_R0010) in response to both IPTG induction and repressor concentration.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1008
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 948
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 662
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