Part:BBa_K2020042

Mj-tRNA with amber anticodon for incorporating ncAA in E.coli

For incorporating unnatural amino acids into a protein, a orthogonal tRNA:Synthetase-pair is needed which does not crossreact with the cognate tRNA:Synthetase-pairs. This tRNA can be assembled with a variety of synthetases into a plasmid to incorporate ncAA in E.coli in response to an amber stop codon

Usage and Biology

This tRNA derives from the wild type tyrosyl Methanococcus janaschii tRNA:Synthetase pair. It was proven to not crossreact with the cognate E.coli tRNA:synthetase-pairs (A Genetically Encoded Photocaged Tyrosine - Schultz et al, 2006).

The tRNA is used together with a tRNA-Synthetase. It has been proven to work with (enter links for parts)

• Y-RS, canonical amino acid
• oNBY-RS
• AzF-synthetase
• CN-F synthetase
• Iodo-Y synthetase
• 5HT-P synthetase
• Nitro-Y synthetase
• Amino-Y synthetase

by [http://2014.igem.org/Team:Austin_Texas iGEM-Team Austin, Texas 2014].

[http://2016.igem.org/Team:Aachen iGEM-Team Aachen 2016] used the tRNA to successfully incorporate canonical amino acid tyrosine with Y-RS, oNB-Y with oNBY-RS and DMNB-S in E.coli BL21 DE3 gold with their newly designed DMNBS-RS.

Incorporation of ncAA

This tRNA has an amber anticodon for incorporating the ncAA in response to an amber codon. It has been used previously in amberless E.coli strain C321.∆A.expb as well as BL21 DE3 gold. When working with a recoded amber codon in BL21 DE3, the ncAA-tRNA is competing with with release factor1 at the amber stop codon. Application of the tRNA is either the incorporation of the ncAA into a protein or usage with a reporter plasmid e.g. pFRY for probing ncAA tRNA/synthetase pair clones regarding efficiency and fidelity.

Assembly in a synthetase plasmid for incorporation of ncAA

pACYC derived plasmid with tRNA and a cognate synthetase

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 the use with a second plasmid, make shure to use replicons from different incompatibility groups, eg. ColE1 and p15A and different selection markers.

Elements of orthogonality

• C1-G72 → most important element for orthogonality. Recognised by Arg174, Arg132, Met178, Lys175 within the synthetase [2]
• A73 → Recognised by Val195 [2]
• G71 → Recognised by Arg132 [2]

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]