Difference between revisions of "Part:BBa K2020045"
| Line 15: | Line 15: | ||
====Assembly in a synthetase plasmid for incorporation of (n)cAA==== | ====Assembly in a synthetase plasmid for incorporation of (n)cAA==== | ||
| − | [[File:T--Aachen-- | + | [[File:T--Aachen--NitroYRS2.png|200px|thumb|left|pACYC derived plasmid with Mj tyrosyl synthetase and cognate tRNA]] |
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 for use with a second plasmid, make shure to use replicons from different incompatibility groups, eg. ColE1 and p15A and different selection markers. A second plasmid could be the [[Part:BBa_K2020040|flourescent reporter plasmid pFRY]] for the purpose of determining fidelity and efficiacy of synthetases for 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 for use with a second plasmid, make shure to use replicons from different incompatibility groups, eg. ColE1 and p15A and different selection markers. A second plasmid could be the [[Part:BBa_K2020040|flourescent reporter plasmid pFRY]] for the purpose of determining fidelity and efficiacy of synthetases for ncAA. | ||
Latest revision as of 00:15, 20 October 2016
NitroY-Synthetase for use in E.coli
This is the 3-Nitro-L-tyrosine-synthetase to be used as a orthogonal synthetase in E.coli. This part can be used together with the cognate tRNA BBa_K2020042 to incorporate NitroY in response to an amber stop codon.
Usage and Biology
Incorporation of ncAA with amber codon supression
Photocleavable non-canonical amino acids offer the opportunity to control protein function on a non-invasive basis. Working with unnatural amino acids requires an additional, orthogonal pair of a tRNA and a cognate synthetase i.e. which does not crossreact with the endogenous tRNA/synthetase pairs [1]. The tRNA's anticodon contains a mutated to amber stop anticodon. Hence, it is possible to incorporate an amino acid at a chosen position in a protein via amber codon suppression.
Assembly in a synthetase plasmid for incorporation of (n)cAA
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 for use with a second plasmid, make shure to use replicons from different incompatibility groups, eg. ColE1 and p15A and different selection markers. A second plasmid could be the flourescent reporter plasmid pFRY for the purpose of determining fidelity and efficiacy of synthetases for ncAA.
Recognition between tRNA and (n)cAA-synthetases derived from Mj
Methanococcus janaschii wild type tyrosyl tRNA consists of two arms: Firstly the acceptor-minihelix, where the amino acid 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 84
Illegal SapI.rc site found at 877