Difference between revisions of "Part:BBa K2020051"

Revision as of 00:00, 20 October 2016

wild type tyrosyl synthetase for use in E.coli with amber anticodon and Y32G

This is a DMNBS-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 tyrosine in response to an amber stop codon. This synthetase has Y32 mutated to glycine and is therefore a template for further mutations for the purpose of changing amino acid specificity.

Usage and Biology

Template for mutations

Site 32 is mutated to glycine, as most evolved synthetases on basis of Mj Y-RS contain this mutation. Former amino acid tyrosine at position 32 is known to build H-bonds with the former substrate tyrosine.

Assembly in a synthetase plasmid for incorporation of ncAA

pACYC derived plasmid with 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 flourescent reporter plasmid pFRY for the purpose of determining fidelity and efficiacy of synthetases for ncAA.

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.

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

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
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 84
Illegal SapI.rc site found at 877

Incorporation of substrates compared to Methanococcus wild type tyrosl synthetase:

Tyrosine %
DMNBS %

@@ Line 9: / Line 9: @@
 ====Template for mutations====
+Site 32 is mutated to glycine, as most evolved synthetases on basis of Mj Y-RS contain this mutation. Former amino acid tyrosine at position 32 is known to build H-bonds with the former substrate tyrosine.
 ====Assembly in a synthetase plasmid for incorporation of ncAA====
@@ Line 16: / Line 16: @@
 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.
+====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.
+====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.