Part:BBa_M36039:Design
Bilirubin Sensor/Actuator Hybrid
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 220
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 220
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 220
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 220
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 220
- 1000COMPATIBLE WITH RFC[1000]
We used GeneDesigner 2.0 to design the plasmid DNA sequence for expression in E. coli. The plasmid contained the genetic code for the UnaG protein. The plasmid contained the following functional characteristics: a kanamycin selection marker, a high copy number which corresponded to roughly 500 copies per cell, a YebF secretion leader, a rhamnose-inducible promoter, and a strong ribosome binding site (RBS). The selection marker was used to allow the plasmid to establish itself within the replicating E. coli cell lineage. Kanamycin was chosen specifically because it was the only marker that was compatible with our chosen secretion leader. We decided to use a high copy number in order to maximize protein production. Bilirubin is a large molecule and is not naturally capable of passing through the cellular membrane. Because of this, we used the YebF secretion leader to secrete the protein construct into the extracellular media where the protein could more readily interact with the bilirubin. The rhamnose-inducible promoter allowed for tunable induction of this secretion activity. The strong RBS was chosen so as increase the affinity of the RBS for the ribosome during translation so as to minimize interference with the mRNA structure during synthesis of the new polypeptide chain.
Source
The gene sequence for the UnaG protein was sourced from the NCBI GenBank database. The original gene was derived from the Japanese eel (Anguilla japonica).