Part:BBa_K567012

tRNA(Asp)-AGG

tRNA(Asp) with its anticodon mutated to CCU(base pairing rare codon AGG) and under the control of aspV promoter. This biobrick is constructed first by cloning the tRNA(Asp) from aspV in E.coli, then the anticodon region is site-directed mutated.

This part is worked coordinately with TDRS(BBa_K567011). TDRS is aspartyl aminoacyl tRNA synthetase without anticodon recognition domain. This modified AspRS can charge Asp to tRNA^Asp-AGG (BBa_K567012). With tRNA^Asp-AGG and TDRS, the ribosome can get through consecutive AGG codons on the mRNA.

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:

tRNA^Asp-AGG (BBa_K567012)

tRNA^Asp-TAG (BBa_K567013)

Get more information from Biobrick

PT7-TDRS (BBa_K567011)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 281
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 281
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 281
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 281
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 281
  
• 1000
  COMPATIBLE WITH RFC[1000]