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, that 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 [3]. The tRNA is used together with a tRNA-Synthetase. It has been proven to work with various synthetases for incorporation of ncAA:

• 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-synthetases:

• DMNBS-RS Clone 1
• DMNBS-RS Clone 2
• DMNBS-RS Clone 3
• DMNBS-RS Clone 4
• DMNBS-RS Clone 5
• DMNBS-RS Clone 6
• DMNBS-RS Clone 7
• DMNBS-RS Clone 8
• DMNBS-RS Clone 9
• DMNBS-RS Clone 10

[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 a recoded amber termination 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. As amber stop codons add up to only about 7% of total stop codos, amber suppression still works appropriately. 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

Fig. 1: 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 [2] and may explain why a variety of ncAA can be incorporated with this tRNA.

Example measurement proving incorporation of amino acids

Fig 2: normalized fluorescence spectrum of mRFP1

Fig 3: normalized fluorescence spectrum of sfGFP as a sign for successful amino acid incorporation via tRNA/synthetase pair for tyrosine via amber supression

Wild Type Methanococcus janaschii tRNA/synthetase pair for incorporation of tyrosine at an amber termination codon - the pair containing this tRNA - is cotransformed with pFRY - Flourescent reporter plasmid for measurement of incorporation of ncAA into BL21 DE3 gold. Reporter plasmid is induced by 100µM ITPG.

pFRY is one part of a two plasmid reporter system for measurement of incorporation of ncAA via amber supression. It consists of two flourescent domains connected through a linker sequence containing and amber stop. When IPTG induced and expressed, the flourescence intensity can be measured. A red flourescence is always visible upon induction, and if an amino acid is incorporated as response to the recoded amber stop codon, then a green flourescence intensity is measurable. Fidelty and efficiacy of the incorporation can be determined with comparison of flourescent level.

This experiment is performed in order to obtain fluorescence spectra of mRFP1 and sfGFP. As you can see in Fig.3 GFP formation was measured as a result from successful amino acid incorporation via amber supression.

Excitation and emission spectra of mRFP1 and sfGFP were obtained from measurement with a modified Biolector set up(Fig 2, Fig 3).

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]