Engineering transcriptional regulators using REPLACE

Transcriptional regulators are fundamental elements in synthetic biology. We sought to use REPLACE to evolve synthetic transcriptional regulators. Through weeks of directed evolution experiments, we succesufuly modified the sensitivity of TetR and PadR to ligand molecules. To evolve PadR, the host cells were initially modified to achieve stable integration of a reporter gene plasmid ((OPadR)6-phCMVmini-PuroR-NLS-mCherry-hPEST-BGHpolyA-pEF1α/HTLV-mTagBFP2-NLS) into the nsP4-expressing BHK-21 cells using lentivirus. The VP64-PadR gene was cloned into the repRNA-v4-derived plasmid. After in vitro transcription, the resulting RNA was delivered into the host cells expressing nsP4 and harboring the stably integrated reporter plasmid through electroporation. Medium was replaced with fresh medium containing 5 μg/mL of puromycin and 10 µg/mL blasticidin 24 h post-electroporation. After 4 d of selection, two million cells were transferred into a new 10-cm culture dish and subjected to consecutive two-day treatment with molnupiravir (10 µM, with daily media changes) to induce RNA mutation. Upon completion of molnupiravir treatment, we adopted different selection/screening strategies according to the specific evolution objectives (i.e., one to evolve ligand-resistant mutants and the other to evolve mutants with enhanced target gene activation in the absence of ligand). To decrease or reverse ligand sensitivity, we employed an evolutionary approach by selecting replicative RNAs containing VP64-PadR with increasing puromycin concentration in the presence of the ligand (200 µM sodium ferulate). In the initial phase of selection, we gradually escalated the concentration of puromycin to inhibit excessive cell growth in the 10-cm culture dish. By enhance the puromycin concentration (up to 60 µg/ml) during mid-screening (e.g., approximately on day 7), around 99% of cells in the dish were effectively eliminated (Approximately 104 to 105 cells in the dish survived in this ‘bottleneck’ stage). This selection was then sustained for another several days. Upon completion of the evolution, cells were collected for RNA extraction and mutation analysis. To enhance the response dynamic range of PadR, we employed an evolutionary approach by selecting replicative RNAs containing VP64-PadR with increasing puromycin concentration in the absence of the ligand (sodium ferulate). We employed a similar approach to gradually enhance the puromycin concentration. However, due to excessive PadR activation activity in the absence of sodium ferulate, we encountered difficulties in eliminating cells by enhancing puromycin concentration (up to 80 µg/ml). Consequently, at the conclusion of the screening period, we utilized flow sorting to isolate approximately the top 1% mCherry-expressing cells (Approximately 105 cells in each dish) from the remaining population for RNA extraction and mutation analysis. To evolve TetR, the host cells were modified to achieve stable integration of a reporter gene plasmid (TRE3G-PuroR-NLS-mCherry-hPEST-BGHpolyA-pEF1α/HTLV-mTagBFP2-NLS) into the nsP4-expressing BHK-21 cells using lentivirus. The TetR-VP48 gene was cloned into the repRNA-v4-derived plasmid. We next employed a similar protocol used in the evolution of PadR to evolve doxycycline-resistant TetR mutants in the presence of 1 µg/ml doxycycline.