Part:BBa_K1831000:Design
NpuDnaE intein for circularization of Type lll AFP+ inker
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
To ensure that the circular AFP would not be strained, and potentially lose function, several linker sequences ranging from 1 to 6 amino acids were modeled using Gromacs. The linkers were developed using a modified version of Team Heidelberg 2014's linker generator software, CRAUT. After the potential linkers were compared in Gromacs, it was determined that adding one glycine to the extein scar least disturbed the ice-binding residues of the AFP and this linker was used in part BBa_K1831000
This construct was designed to circularize any linear protein, however part BBa_K1362000 from which is was based lacked a T7 promoter. Therefore sub-cloning of the construct would be required by other teams prior to expression of their circular protein. QGEM 2015 was able to add a constitutive T7 promoter, part BBa_I14018, to simplify the use of the NpuDnaE intein for future teams.
Source
This part was derived from BBa_K1362000, a composite part submitted by Team Heidelberg 2014's NpuDnaE intein RFC[105] circularization construct. We modified the part by adding part BBa_I14018, a constitutive T7 promoter with a medium transcription level prior to the RBS.