GFP (E0040) with two amber stop codons
This part encodes the standard registry GFP (BBa_E0040), strong promoter/RBS pair (BBa_BBa_J23038), and terminator (BBa_B0010). However, this part will not express without the supP tRNA submitted by Stanford-Brown-Spelman 2014 (BBa_K1499251).
Usage and Biology
This part is GFP with two inserted TAG stop codons in place of leucine residues. This part transformed into Amberless cells along with supP tRNA will express GFP. In wild type cells, no GFP will be expressed as the TAG will be recognized as stop codons rather than leucine. This part is used as a proof of concept for Codon Security as part of the Amberless Hell Cell project which mitigates the effects of horizontal gene transfer.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 705
The part was sequence verified in the pSB1C3 backbone before submission to the registry. One forward read was obtained using VF2.
This part was verified to work in conjunction with the supP tRNA (Figure 3). As seen, the part by itself does not express GFP because of the stop codons.
The system with the supP tRNA works significantly better in amberless E. coli compared to DH5-alpha (Figure 4). Again, in both amberless and DH5-alpha cells, there is no GFP fluorescence due to the termination at the stop codon.