Part:BBa_K2020051

wild type tyrosyl synthetase for use in E.coli with amber anticodon and Y32G

This is a DMNBS-synthetase to be used as a orthogonal synthetase in E.coli. This part can be used together with the cognate tRNA BBa_K2020042 to incorporate tyrosine in response to an amber stop codon. This synthetase has Y32 mutated to glycine and is therefore a template for further mutations for the purpose of changing amino acid specificity.

Usage and Biology

Assembly in a synthetase plasmid for incorporation of ncAA

[[File:|200px|thumb|left|pACYC derived plasmid with tyrosyl-synthetase and cognate tRNA]]

Most synthetases are used with low copy plasmids (e.g. pACYC). Assemble the tRNA and the synthetase into a low copy plasmid, each one with an own promoter and one terminator for both. (See picture). If your application is not for incorporation into a protein but for use with a second plasmid, make shure to use replicons from different incompatibility groups, eg. ColE1 and p15A and different selection markers. A second plasmid could be the flourescent reporter plasmid pFRY for the purpose of determining fidelity and efficiacy of synthetases for ncAA.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 84
  Illegal SapI.rc site found at 877
  

Incorporation of substrates compared to Methanococcus wild type tyrosl synthetase:

• Tyrosine %
• DMNBS %