Difference between revisions of "Part:BBa K2020042"
(→Usage and Biology) |
(→Usage and Biology) |
||
| Line 32: | Line 32: | ||
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. | ||
| + | |||
| + | ====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 | ||
<!-- --> | <!-- --> | ||
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)
- 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
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]