Difference between revisions of "Part:BBa K2020051"
(→‎Assembly in a synthetase plasmid for incorporation of ncAA)
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====Assembly in a synthetase plasmid for incorporation of ncAA====
 
====Assembly in a synthetase plasmid for incorporation of ncAA====
  
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[[File:T--Aachen--Mj YRS CUA.jpg|200px|thumb|left|pACYC derived plasmid with Mj tyrosyl synthetase and cognate tRNA]]
+
[[File:|200px|thumb|left|pACYC derived plasmid with DMNBS-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.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
−
<partinfo>BBa_K2020051 SequenceAndFeatures</partinfo>
 
  
 +
<partinfo>BBa_K2020051 SequenceAndFeatures</partinfo>
 +
Incorporation of substrates compared to Methanococcus wild type tyrosl synthetase:
 +
*Tyrosine %
 +
*DMNBS %
  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2020051 parameters</partinfo>
 
<partinfo>BBa_K2020051 parameters</partinfo>

Revision as of 09:26, 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 DMNBS in response to an amber stop codon.


Usage and Biology

Incorporation of DMNBS

Assembly in a synthetase plasmid for incorporation of ncAA

[[File:|200px|thumb|left|pACYC derived plasmid with DMNBS-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


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 %

Functional Parameters