Difference between revisions of "Part:BBa K2020051"
(→Assembly in a synthetase plasmid for incorporation of ncAA) |
|||
| Line 3: | Line 3: | ||
<partinfo>BBa_K2020051 short</partinfo> | <partinfo>BBa_K2020051 short</partinfo> | ||
| − | 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 | + | 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=== | ===Usage and Biology=== | ||
| − | ==== | + | ====Template for mutations==== |
====Assembly in a synthetase plasmid for incorporation of ncAA==== | ====Assembly in a synthetase plasmid for incorporation of ncAA==== | ||
| − | [[File:|200px|thumb|left|pACYC derived plasmid with | + | [[File:|200px|thumb|left|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 [[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. | ||
Revision as of 21:20, 15 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
Assembly in a synthetase plasmid for incorporation of ncAA
[[File:|200px|thumb|left|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.
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
Incorporation of substrates compared to Methanococcus wild type tyrosl synthetase:
- Tyrosine %
- DMNBS %