Part:BBa_K567011

PT7-TDRS

aspartyl aminoacyl tRNA synthetase without anticodon recognition domain under the control of T7 promoter and lac operator. This biobrick is constructed by deleting the anticodon recognition domain of AspRS from E.coli. This modified AspRS can charge Asp to tRNA(Asp)-TAG (BBa_K567013) and tRNA(Asp)-AGG (BBa_K567012).

To deprive AspRS of its anticodon specificity, we analyzed the structure of AspRS and expressed a truncated AspRS without anticodon recognition domain. Our design is shown below. This modified enzyme keeps its ability of aminoacylation while loses its activity of recognizing anticodon of tRNA.

Fig 1. The design for modification is based on the crystal structure of E.coli AspRS coupled with tRNA (PDB ID: 1C0A). This figure shows E.coli aspartyl-tRNA synthetase with and without anticodon recognition domain. After this domain is depleted, the enzyme cannot bind tRNA^Asp anticodon. Yet the ability of aminoacylation is maintained.

We have used PT7-RFP-6AGG (BBa_K567017) as our Reporter. We have constructed tRNA^Asp-AGG and PT7-TDRS (AspRS without anticodon recognition domain, ) (BBa_K567012 and BBa_K567011)as the Modulator. tRNA^Asp-AGG, which can recognize rare codon AGG, is under constitutive promoter. Our device works as follows: Without Modulator, RFP with 6 consecutive AGG insertions can hardly be expressed. When Modulator works, tRNA^Asp-AGG can be charged with Asp by TDRS. tRNA^Asp-AGG recognizes rare codon AGG on the Reporter RFP-6AGG. The ribosome gets through this part of the mRNA more easily. RFP is expressed. Red fluorescence is observed. Our experiment results are shown below.

Fig 2. With our Modulator (BBa_K567012 and BBa_K567011), Reporter RFP-6AGG is expressed, demonstrating that our system works well.

Fig 3. aaRS Modulator + Reporter for Qualitative Analysis. aaRS Modulator + RFP-6AGG(the middle three) emit red fluorescence. Wild type RFP (the first one from the left) exhibits bright red fluorescenceis. Control (first one from the right) exhibits no red fluoresence.

Related Biobrick: BBa_K567012BBa_K567013

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1595
  Illegal EcoRI site found at 1767
  Illegal XbaI site found at 48
  Illegal PstI site found at 1152
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1595
  Illegal EcoRI site found at 1767
  Illegal NheI site found at 1556
  Illegal PstI site found at 1152
  Illegal NotI site found at 1620
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1595
  Illegal EcoRI site found at 1767
  Illegal BamHI site found at 1589
  Illegal XhoI site found at 1629
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1595
  Illegal EcoRI site found at 1767
  Illegal XbaI site found at 48
  Illegal PstI site found at 1152
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1595
  Illegal EcoRI site found at 1767
  Illegal XbaI site found at 48
  Illegal PstI site found at 1152
  
• 1000
  COMPATIBLE WITH RFC[1000]