Successful in vivo directed evolution by PREDCEL and PACE requires the thorough consideration of experimental parameters, e.g. phage propagation times, culture dilution rates and inducer/inhibitor concentrations. We employed extensive ODE-based and stochastic modeling to identify the most sensitive parameters and adapt our experiments accordingly. First, we calibrated our models using phage propagation experiments from our wet lab complemented with literature data. Simulations showed that the phage titer is highly sensitive to culture dilution rates. We simulated batch times and transfer volumes for PREDCEL and corresponding flow rates for PACE to determine optimized conditions for gene pool selection while avoiding phage washout. We also estimated phage titer monitoring intervals for cost/labor efficient QC as well as inducer/inhibitor concentrations required to express the required mutagenic polymerases. Finally, provide a web-based, fully interactive modeling platform, not only extensively employed by our wet lab, but highly informs future iGEM teams building on our work.
Phage titer
Simulations of phage and E. coli titer support both PREDCEL and PACE by helping to choose a set of experimental parameters that is both efficient in terms of directed evolution and in terms of usability.
Use the interactive tools to simulate the conditions you are interested in and explore how the combined experimental parameters influence experimental outcomes.
Model the glucose and arabinose concentration to make sure mutagenesis plasmids are sufficiently induced to get optimal mutagenesis conditions for both PREDCEL and PACE.
Estimate the number of mutated sequences in a PREDCEL or PACE experiment at a given point in time to check for the covered sequence space and to save time and mony when sequencing.
