Part:BBa_K4339001

Alanine tRNA synthetase

Alanine tRNA synthetase is an enzyme that catalyses the binding of alanine to tRNA. Modelling demonstrated that the expression of spider silk proteins (spidroins), which have a number of repetitive alanine regions, would be increased with the co-expression of alanine tRNA synthetase.

Usage and Biology

Sequence and Features

The sequence originates from E. coli MG1655 K-12 (Puteny et al. 1981)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2066
  Illegal PstI site found at 2582
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2066
  Illegal PstI site found at 2582
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 629
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2066
  Illegal PstI site found at 2582
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2066
  Illegal PstI site found at 2582
  Illegal AgeI site found at 709
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1090
  Illegal SapI.rc site found at 1138
  Illegal SapI.rc site found at 1288
  

The enzyme was intended for co-expression with MaSps (Major Ampullate Spidroin Proteins) as part of a helper plasmid, containing genes for alanine tRNA and CycA (part K4339000 - an alanine import channel protein) expression. This system was designed to increase alanine uptake and synthesis of alanyl-tRNA to maximise possible rate of alanine incorporation into protein and thus, production of alanine-rich MaSps.

This part was successfully synthesised but was not transformed due to time constraints.

References

Putney S et al. Purification and properties of alanine tRNA synthetase from Escherichia coli. A tetramer of identical subunits. Journal of Biological Chemistry. 1981;256(1): 198–204. doi: https://doi.org/10.1016/s0021-9258(19)70119-7