Part:BBa_K1499252

GFP with 2 stop codons generator + supP tRNA

This composite part combines an ambered GFP generator (K1499250, 2 amber stop codons) with the amber-suppressing tRNA supP (mutated LeuX, K1499251).

Usage and Biology

This construct is used to validate our amberless expression system, an orthogonal translation system that uses an amber-suppressing tRNA to incorporate leucine into proteins at sites coded for by the amber stop codon (UAG). This system works to mitigate the effects of horizontal gene transfer by ensuring proper expression only in cells a) carrying the tRNA and b) lacking the release factor responsible for ending translation at a UAG (RF1). We have combined our GFP construct (which differs from E0040 at only two sites, changing normal leucine codons to UAGs) with a well-categorized constitutive promoter (J23104), RBS (B0034), and terminator (B0010). Added to the end of these four parts is the supP tRNA, from Stanford-Brown-Spelman's 2014 Amberless toolkit, or BBa_K1499251.

Figure 1. Our approach to the Amberless Codon Security. 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.

The complete generator was used to test the orthogonality of the UAG->Leucine coding (Figure 2).

Figure 2. We transformed DH5-alpha and amberless cells with test plasmids, in this case the GFP reporter gene with stop codons and the supP tRNA, in order to establish a proof-of-concept for orthogonality using Codon Security.

We have observed that this generator works well in amberless E. coli, but not DH5-alpha. Because of the toxicity of the tRNA, the part will be difficult to transform into any strain of bacterium that contains genes with the amber stop 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 (Figure 3).

Figure 3. GFP with UAG stop codons and supP tRNA match expected sequence. The GFP goes from bp 167 to 888.The tRNA goes from bp 1141 to 1225.

Results

This part successfully expressed GFP in Amberless cells but not in DH5-alpha (Figure 4).

Figure 4. Flow cytometry histogram showing that Amberless cells with the GFP-2S+tRNA construct strongly express GFP, while DH5-alpha cells have a mixed population of low-expressing and non-expressing cells.

The system works significantly better in amberless E. coli compared to DH5-alpha (Figure 5).

Figure 5.Mean fluorescence from flow cytometry data in Figure 4. The data show a high mean fluorescence in amberless cells and a much lower mean fluorescence in DH5-alpha cells containing the same construct.