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Coding
ONBY RS

Part:BBa_K1416000

Designed by: Jordan Monk   Group: iGEM14_Austin_Texas   (2014-10-06)

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 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 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 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
    COMPATIBLE WITH RFC[1000]


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