Part:BBa_K5005001:Design
EGFP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
EGFP is a green fluorescent protein that can be constructed onto the pTarget plasmid. A point mutation at the 199th amino acid of EGFP can cause the protein to transform into the blue fluorescent protein. Conversely, a mutation (C-G to T-A) in EBFP can also convert it back into EGFP. Thus, by conducting experiments in which EBFP is converted back into EGFP, we can demonstrate the practicality of the TRACE system.
Target plasmid with EGFP located downstream of the T7 promoter. EGFP can be converted into BFP through site-directed mutagenesis, which changes one amino acid. This eGFP serves as a template for our transformation, using point mutations to mutate it into the BFP we need to change green fluorescence into blue fluorescence.
ggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacg is the forward primer of eGFP2BFP-F.tagcggctgaagcactgcacgccgtgggtcagggtggtcacga is the reverse primer of eGFP2BFP-R.These two primers are used for point mutation of eGFP which helps turn eGFP into BFP.
Source
Constructed in this work.
References
Chen, H., Liu, S., Padula, S., Lesman, D., Griswold, K., Lin, A., Zhao, T., Marshall, J. L., & Chen, F. (2020). Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nature biotechnology, 38(2), 165–168. https://doi.org/10.1038/s41587-019-0331-8.