Part:BBa_K4942007
EGFP
GFP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Modification and Applications by NNHS 2024
NNHS 2024: We utilized the EGFP in our project as a key indicator of mutagenesis. In our project, we designed a fusion protein incorporating rAPOBEC1, a cytidine base editor (CBE), and T7 RNA polymerase that served as the main mutagenesis tool.
As the RNA polymerase brings the protein alongside the DNA sequence starting at the T7 promoter, the CBE has the ability of conducting mutagenesis, converting cytosine bases into thymine. In addition, the components of n-Mag and p-Mag split our “black box” protein into two parts, only to be activated under blue light. While it is rather difficult to confirm mutagenesis on other proteins, using EGFP is especially convenient as there would be an obvious change in the overall fluorescence level, either in the negative or positive direction.
In experimentation, compelling evidence is seen on the effects of our “black box” protein on EGFP mutagenesis. In the fluorescence microscopy data, EGFP is evidently brighter as a result of the use of the CBE-RNAP fusion protein. The congregation abilities of the EGFP likely have improved. In further quantification, it is seen that the fluorescence level has generally increased with statistical significance. Meanwhile, the proteins exhibiting decreases in fluorescence prove that mutagenesis is not one sided.
Using Sanger sequencing, we analyzed the amino acid sequences of the mutant EGFP, which was then predicted for protein folding and then visualized. Indeed, these mutations provide reason for the increased fluorescence levels. The mutation at position 29 from serine to phenylalanine saw the addition of a benzene ring, which likely allowed the EGFP to better bind with other EGFP proteins.
Overall, the use of EGFP is greatly effective as a green light for mutagenesis. Even when we are attempting to optimize other proteins involved in the synthesis of spermidine and NADPH or the degradation of nicotinate, we used the extent to which fluorescence level from the EGFP has changed to prove that we have been shining our system with enough blue light to actually allow the mutagenesis of the important enzymes that we are focusing on.
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