Part:BBa_K3143010
Phd3TAG-GS linker-sfGFP
Phd3TAG-GS linker-sfGFP is used to quantify the effect of TAG codon insertion into Phd antitoxin. By doing so, we can simply characterize the performance of Phd(nTAG) by measuring fluorescence. See more about see Phd-G4S-sfGFP BBa_K3143007, Phd2TAG-G4S-sfGFP BBa_K3143008 and Phd3TAG-G4S-sfGFP BBa_K3143009.
Characterization
To better quantify the effect of insertion the TAG codon into Phd antitoxin, we first fused a GFP fluorescent protein using the GS Linker to the C-terminus of the PhD protein. By doing so, we can simply characterize the performance of the TAG codon insertion by measuring fluorescence.
Figure 1: Schematic illustration of genetic circuits for incorporation of ncAAs into Phd antitoxin.
First we tried to confirm that insertion of how many TAG codons could completely inhibit gene expression. We inserted 1-3 TAG codons just after the start codon, and then measured the fluorescence value of Phd-GS-sfGFP. The results showed the expression of the gene was well terminated in three designs, and the insertion of two TAG codons was the best.
Figure 2: Characterization of Phd-TAG-GS-sfGFP system
Next we attempted to introduce the ncAA system into the Phd-GS-sfGFP expression system with two TAG codons. When the ncAA system is expressed, we can see that the system that originally had no fluorescence produced a strong fluorescent signal. This shows that we have successfully established an Anticoxin (Phd) system that relies on ncAA. When ncAA is present in the system, Phd can be successfully expressed, and when ncAA is not present in the system, the expression of Phd is completely terminated.
Figure 3: Characterization of Phd(2TAG)-Doc system
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 250
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