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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1153
Illegal BamHI site found at 1159 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 323
Illegal NgoMIV site found at 1185
Illegal NgoMIV site found at 1645 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 316
Illegal SapI.rc site found at 1109
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