Difference between revisions of "Part:BBa K2020042"

(Usage and Biology)
(Usage and Biology)
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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.
 
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.
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====Elements of orthogonality====
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*C1-G72 → most important element for orthogonality. Recognised by Arg174, Arg132, Met178, Lys175 within the synthetase
 +
*A73 → Recognised by Val195
 +
*G71 → Recognised by Arg132
  
 
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Revision as of 10:37, 16 October 2016


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)

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
  • A73 → Recognised by Val195
  • G71 → Recognised by Arg132

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]

Functional Parameters