Part:BBa_K1416000

The tRNA synthetase/tRNA needed for incorporating o-(2-nitrobenzyl)-L-tyrosine (ONBY) at a UAG codon

The part includes the gene coding for the Methanocaldococcus jannaschii synthetase mutated to charge the orthogonal tRNA (also in the part) with the non-canonical amino acid o-nitrobenzyl tyrosine (ONBY) along with the proper promoters and terminator. This part encodes for the incorporation of ONBY during translation at AUG amber stop codons in a cell with Release Factor 1 (RF1) knocked out such as "Amberless" E. coli or "RF0" E. coli.

This part was used by the UT Austin 2014 iGEM team in their [http://2014.igem.org/Team:Austin_Texas/kit#Results_and_Data ncAA kit project]. In this work they demonstrated that while ONBY itself is slightly toxic to the cells, the synthetase/tRNA pair acted with good fidelity at incorporating only ONBY. Additionally, this part was also used in the UT Austin 2014 iGEM [http://2014.igem.org/Team:Austin_Texas/photocage Photocage project], where the part successfully incorporated ONBY within T7 RNA polymerase (RNAP), yielding a light-activated T7 RNAP.

Data showing the characterization of BBa_K1416000. In the control cultures, ONBY-pFRYC, the level of GFP expression relative to RFP expression is set as a standard. In the test cultures, ONBY-pFRY, the level of GFP expression relative to RFP expression indicates how well the part function. In the absence of ncAA, there is virtually no GFP expression relative to RFP expression. Compared to the control, this indicates that the BBa_K1416000 has high fidelity. In the presence of ncAA, the amount of GFP expression increases notably. These results indicate that while BBa_K1416000 has high fidelity, it is only moderately efficient. For more information and details, please visit [http://2014.igem.org/Team:Austin_Texas/kit#Results_and_Data ncAA kit project]

Sequence and Features

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 323
  Illegal NgoMIV site found at 1185
  Illegal NgoMIV site found at 1645
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 316
  Illegal SapI.rc site found at 1109