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 various synthetases for incorporation of ncAA:

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.

[http://2016.igem.org/Team:TU_Darmstadt iGEM-Team TU Darmstadt 2016] worked with OMeY-synthetase with this tRNA.

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]

Recognition between tRNA and ncAA-synthetases

Methanococcus janaschii wild type tyrosyl tRNA consists of two arms: Firstly the acceptor-minihelix, where the ncAA will be attached to the 3' end. Secondly the anticodon containg arm. Synthetases interact mainly with the acceptor minihelix of the tRNA. Due to the lack of most of a recognizing element within the anticodon containg section, a mutation of a anticodon base has a relatively small effect on the aminoacylation efficiency [1] and may explain why a variety of ncAA can be incorporated with this tRNA.

Sequence and Features

Assembly Compatibility: