Part:BBa_K567013

tRNA(Asp)-TAG

tRNA(Asp) with its anticodon mutated to CUA (base pairing stop codon UAG) 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.

Construction of BBa_K567013

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 constructed on the backbone plasmid pACYC184.

Characterization of BBa_K567013

This part acts as a stop codon suppressor tRNA. It can be charged with Asp.

We have used Pbla-Luc-TAG (BBa_K567003) as our Reporter. The amount of luciferase produced is reflected using the bioluminescence emitted during the luciferin reaction. Our results demonstrate that TAG insertion into luciferase blocks luciferase production, which was shown in the control group. In the experimental group, with the help of PT7-TDRS(BBa_K567011) and tRNA^Asp-TAG(BBa_K567013),luciferase was produced and bioluminescence was emitted during the luciferin reaction. These results proved that Stop-Codon Switch can turn on protein expression. Yet further work is needed to optimize the device.

Fig.1 Functional Analysis of Stop-Codon Switch. ER2566 cannot produce luciferase with Pbla-Luc-TAG (BBa_K567003) only. When PT7-TDRS (BBa_K567011) and tRNA^Asp-TAG (BBa_K567013) are also transformed into the cell, luciferase is produced. The results proved Stop-Codon Switch as a strict molecular switch without background noise.

Related 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]