Part:BBa_K3590040
Scaffoldin for degradation of microcystin - MC-LR
ScafD was designed to display enzymes MlrA, B and C from the microcystin degradation pathway. Thanks to the close proximity of those enzymes, displayed on this scaffoldin, microcystin degradation will be more efficient compared to freely released enzymes.
At the 5' end of ScafD there is the Pveg promoter followed by RBS R2. The CDS encodes several functional modules - SacB signal sequence for extracellular transport, His-tag for Western blot detection followed by the MlrA enzyme that linearizes the circular molecule of MC-LR. The MlrA module is connected by a linker to a cohesin molecule that originates from Bacteroides cellulosolvens which is followed by another linker and cohesin from Acetivibrio cellulolyticus. Those cohesin domains ensure the attachment of MlrB and MlrC enzymes to the ScafD, respectively. The attachment of these enzymes is achieved through a species-specific interaction of cohesins and their counterparts - dockerins, which are fused with said enzymes. Another linker then connects the previous parts of the fusion protein to three LysM domains that anchor the whole system to peptidoglycans in the cell wall of B. subtilis. At the 3' end of the CDS, there is a STOP codon.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1612
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1612
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1612
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1612
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2811
Illegal BsaI.rc site found at 306
//chassis/prokaryote/bsubtilis
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