Difference between revisions of "Part:BBa K2201200:Design"

 
 
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
Remove the TyrRS and replace with the 2-NPA-RS.
+
We created this specific aaRS by ordering a gene synthesis at Integrated DNA Technologies (IDT) with a codon optimized sequence of an aaRS able to incorporate 2-NPA, based on an evolution experiment of Peters et al. This gene synthesis was integrated into the ONBY-Part K1416000, replacing the ONBY-RS. This approach is applicable, as the 2-NPA-RS and the ONBY-RS are mutated versions of the <i>M. jannaschii</i> TyrRS, such that both recognize the same tRNA and only the sequence of the aaRS itself has to be changed to get a new synthetase binding other amino acids to the tRNA. The gene synthesis we ordered had 30 base pairs overlap to a linearized vector of the ONBY-part K1416000, where only the gene sequence of the ONBY-RS was removed. The fragment of the NPA-RS was integrated by Gibson assembly (<b>Figure 1</b>.
  
 +
[[File:T--Bielefeld-CeBiTec--YKE_P7_NPA_RS_Construction.png|thumb|600px|center| <b>Figure 1:</b> History of the plasmid construction of K2201200.]]
  
  

Latest revision as of 14:34, 1 November 2017


Tyrosyl tRNA/aminoacyl-synthetase for the incorporation of 2-nitrophenylalanine


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1153
    Illegal BamHI site found at 1159
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 404
    Illegal NgoMIV site found at 1185
    Illegal NgoMIV site found at 1645
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We created this specific aaRS by ordering a gene synthesis at Integrated DNA Technologies (IDT) with a codon optimized sequence of an aaRS able to incorporate 2-NPA, based on an evolution experiment of Peters et al. This gene synthesis was integrated into the ONBY-Part K1416000, replacing the ONBY-RS. This approach is applicable, as the 2-NPA-RS and the ONBY-RS are mutated versions of the M. jannaschii TyrRS, such that both recognize the same tRNA and only the sequence of the aaRS itself has to be changed to get a new synthetase binding other amino acids to the tRNA. The gene synthesis we ordered had 30 base pairs overlap to a linearized vector of the ONBY-part K1416000, where only the gene sequence of the ONBY-RS was removed. The fragment of the NPA-RS was integrated by Gibson assembly (Figure 1.

Figure 1: History of the plasmid construction of K2201200.


Source

Standard M.jannaschii TyrRS plasmid.

References