Difference between revisions of "Part:BBa K2020042"

(→‎Usage and Biology)
(→‎Incorporation of ncAA)
Line 23: Line 23:
  
 
====Incorporation of ncAA====
 
====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. Application of the tRNA is either the incorporation of the ncAA into a protein or usage with a reporter plasmid e.g. [[Part:BBa_K2020040|pFRY]].
+
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. Application of the tRNA is either the incorporation of the ncAA into a protein or usage with a reporter plasmid e.g. [[Part:BBa_K2020050|pFRY]].
  
 
====Assembly in a synthetase plasmid for incorporation of ncAA====
 
====Assembly in a synthetase plasmid for incorporation of ncAA====

Revision as of 21:08, 14 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)

  • Y-RS, canonical amino acid
  • oNBY-RS
  • AzF
  • 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 tyrosine, oNB-Y 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. Application of the tRNA is either the incorporation of the ncAA into a protein or usage with a reporter plasmid e.g. pFRY.

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.

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