Coding

Part:BBa_K1499250

Designed by: Raman Nelakanti   Group: iGEM14_StanfordBrownSpelman   (2014-08-27)

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.

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.

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

Characterization

Verification

The part was sequence verified in the pSB1C3 backbone before submission to the registry. One forward read was obtained using VF2.

Figure 2. GFP with two amber stop codons matches the expected sequence.

Results

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.

Figure 3. 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

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.

Figure 4. 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


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