Difference between revisions of "Part:BBa K2020043"

 
Line 5: Line 5:
 
This is the 4-Azido-L-phenylalanine-synthetase to be used as a orthogonal synthetase in E.coli. This part can be used together with the [[Part:BBa_K2020042|cognate tRNA BBa_K2020042]] to incorporate AzF in response to an amber stop codon.  
 
This is the 4-Azido-L-phenylalanine-synthetase to be used as a orthogonal synthetase in E.coli. This part can be used together with the [[Part:BBa_K2020042|cognate tRNA BBa_K2020042]] to incorporate AzF in response to an amber stop codon.  
  
−
<!-- Add more about the biology of this part here
+
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
 +
 +
====Incorporation of AzF====
 +
 +
 +
 +
====Assembly in a synthetase plasmid for incorporation of ncAA====
 +
 +
[[File:T--Aachen--AzF.jpg|200px|thumb|left|pACYC derived plasmid with AzF-synthetase and cognate tRNA]]
 +
 +
Most synthetases are used with low copy plasmids (e.g. pACYC). Assemble the [[Part:BBa_K2020042|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 21:36, 14 October 2016


AzF-Synthetase for use in E.coli

This is the 4-Azido-L-phenylalanine-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 AzF in response to an amber stop codon.


Usage and Biology

Incorporation of AzF

Assembly in a synthetase plasmid for incorporation of ncAA

pACYC derived plasmid with AzF-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