Part:BBa_K584019

OmpA_transmembrane_domain + Linker + point_mutant_HPLC6(AFP)

This composite part contains the two sequences OmpA and Point Mutant HPLC6(Antifreeze Protein - AFP) that are linked via a flexible linker.

AFP normally is expressed intracellularly to prevent freezing of the cytoplasm, but in our system, it was essential that AFP was extracellularly anchored at the cell membrane, thereby preventing ice formation outside the cell. Therefore, we merged the AFP gene to OmpA via a flexible linker, thereby ensuring the desired extracellular localization of AFP.

A point mutation at Base Pair number 156 of the HPLC6 sequence was performed by replacing the T in the wild-type coding sequence to C.This mutation was necessary as the sequence CTGCAG in the Wild-type coding sequence corresponded to a Pst1 restriction site. GCT(Wild type) and GCC(1 point mutant) are both valid triplet codons for Alanine.

Detailed information of the Pseudopleuronectes americanus Antifreeze Protein (HPLC gene) can be found on this NCBI webpage [http://www.ncbi.nlm.nih.gov/nuccore/M62417.1]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]