Part:BBa_K2910003:Design
IsPETase-T88F-I208Y-S238F-S245Y-R280A with C-terminal Hexahistidine Tag
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 67
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Two computational tools were used to inform the rational design process: FlexddG, a Rosetta protein design algorithm which can take backbone flexibility into account, and which gives an accurate prediction of the energy change after mutation; and FuncLib, a web server which can quickly and exhaustively screen combinations of mutants.
FlexddG was used to test substitutions at each candidate position. These were selected on a rational basis, to better interact with local motifs on the PET ligand. The best performers were fed into FuncLib; the most commonly substituted amino acids in the top 600 sequences as scored by FuncLib were used to put together a short-list of multiple mutants, which were tested using FlexddG again. This sequence was a Top 4 performer in this final testing process.
A detailed description of this process is available on the iGEM Toronto 2019 Wiki.
Source
This part came from the PDB crystal structure 5XJH fed into the algorithm above, followed by back-translation into DNA sequence using EMBOSS-Backtranseq.