Blue-shifting green fluorescent proteins using REPLACE
In the directed evolution experiments of EGFP or StayGold, the objective was to blue-shift the excitation light spectrum of GFP towards shorter wavelengths, resulting in an enhancement of fluorescence intensity upon 405 nm laser excitation. This facilitates the advancement of novel fluorescent proteins. These experiments represent a category of experiments that utilize REPLACE to achieve discontinuous directed evolution through FACS sorting (screening). Overall design: EGFP or StayGold was cloned into the repRNA-v4-derived vector. The vector was in vitro transcribed into RNA and delivered into host cells via electroporation. After 24 hours of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages. After another 5-7 days, a relatively stable and homogeneous population of polyclonal cells carrying replicative RNA with expression of EGFP or StayGold was generated (i.e., Parent). Approximately 1-2×106 cells were passaged into a new 10-cm culture dish and treated with molnupiravir (10 µM) for two consecutive days with daily media changes. Then, they were divided equally into five separate 10-cm dishes and subjected to flow cytometry sorting after an additional cultivation period of approximately 3-4 days. The sorting process involved drawing a triangular region along the diagonal and applying a gate to approximately 0.5% positive cells of the cell population. About 104 cells were sorted and cultured as positive population (i.e., Post_1st). After about 7 days, these cells are expected to proliferate to a quantity of around 107. Similarly, these cells were exposed to a two-day treatment with molnupiravir (10 µM) and subsequently transferred onto five new 10-cm dishes for cultivation over a period of 3-4 days. The positive population was then sorted using flow cytometry at a ratio of 0.5%. The sorted cells were cultured to a count of approximately 106 and utilized for cryopreservation and total RNA isolation (i.e., Post_2nd). The extracted RNA was reverse transcribed into cDNA and then utilized for mutation analysis and mutant identification.