Coding

Part:BBa_K3900000:Design

Designed by: Lennart Lutz   Group: iGEM21_Bielefeld-CeBiTec   (2021-09-21)


Ribose Binding Protein NNK Sequence for Darwin-Assembly


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 385
    Illegal PstI site found at 601
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 385
    Illegal PstI site found at 601
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 385
    Illegal PstI site found at 601
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 385
    Illegal PstI site found at 601
    Illegal NgoMIV site found at 480
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

While designing the part the main decisions we had to make were which triplets to modify with NNK-primers. Different amino-acids in the binding pocket were possible candidates but to keep the amount of mutations realistic we chose to mutate the six most relevant.

The decision process was supported by evaluating 3D-structures of the receptor. The amino acids were determined by analyzing structure predictions in PyMol after the chemicals Diisopropyl methylphosphonate (DIMP) and Benzenetricarboxylic acid (BTCA) were docked using Rosetta. If an amino acid was found suitable for mutation depended on the interactions to the ligand already occurring and the distance between ligand and amino acid.

Afterwards primers with NNK-triplets in the middle were designed.




Source

The NNK-RBP-sequence is based on the sequence of the ribose binding protein from Escherichia coli. The DNA-sequence was taken from https://www.rcsb.org/structure/1URP .

References