Part:BBa_K1499250

GFP (E0040) with two amber stop codons

This part encodes the standard

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.

Figure 1. Our approach to the Amberless Hell Cell. By recoding the UAG stop codon to translate into an amino acid, only cells that have a tRNA with the anticodon AUC will produce the complete protein. In our experiment, we used a tRNA that charges with leucine to translate the UAG codon.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 705
  

Verification

The part was sequence verified in the pSB1C3 backbone before submission to the registry. Two reads, forward and reverse, were obtained using VF2 and VR.

[[File:|700px|thumb|center|Figure 2. T GFP with two amber stop codons matches the expected sequence. ]]

Results

Figure 2. The use of this part can be shown in the right most graph. No GFP expression is observed as the stop codon truncates the protein. In the Amberless cells, when accompanied with the supP tRNA, this part does lead to the expression of GFP

Figure 2. The use of this part can be shown in the wild_type GFP bar. No GFP expression is observed as the stop codon truncates the protein. In the Amberless cells, when accompanied with the supP tRNA, this part does lead to the expression of GFP