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.

Reporter:We test our design with two reporters.

RFP-6AGG (BBa_K567017) : RFP with 6AGG insertions

Luciferase-4AGG (BBa_K567009) : luciferase with 4AGG insertions

When the modified enzyme is produced under induction, it can charge tRNA^Asp-AGG with Arg. Then the charged tRNA can get through the rare codons on the mRNA, so that RFP or luciferase can be produced.

File:11SJTU rare 03.jpg

Fig.1 With our device (BBa_K567012 and BBa_K567011), RFP-6AGG is expressed.

Fig.2 Red fluorescence can be observed in cells with our device (the middle three). Wild type RFP (the first one from the left) exhibits bright red fluorescenceis. Control (first one from the right) exhibits no red fluoresence.

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). tRNA^Asp-AGG, which can recognize rare codon AGG, is under constitutive promoter. With our device, RFP is successfully produced. Without our device, little RFP is observed.

Fig.3 Examination of luciferase production with and without device. ER2566 cannot produce luciferase with PT7-Luc-4AGG (BBa_K567009) only. When tRNA^Asp-AGG (BBa_K567012) and PT7-TDRS (BBa_K567011) are co-transformed into the cell, luciferase production is increased. The results proved that aaRS can regulate protein biosynthesis.

We have used PT7-Luc-4AGG (BBa_K567009) as our Reporter to test the function of PT7-TDRS (BBa_K567011) and tRNA^Asp-AGG (BBa_K567012). Results are shown above. Luciferase production has been largely increased with our device.

The function of Switch is characterized by the amount of luciferase expressed. The amount of luciferase expressed is reflected by the light emitted when luciferase acts on the appropriate luciferin substrate. The light can be measured by luminometer and the quantity is positively correlated with the amount of luciferase and its activity ([http://2011.igem.org/Team:SJTU-BioX-Shanghai/Notebook/Protocol learn more...]).

We successfully controlled protein expression by manipulating aaRS.

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]