Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of protocols for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, by detailing the approach to protocol development, these methods are also adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.
Keywords: 2-Stage; Dynamic control; E. coli; High throughput; Metabolic engineering; Microbial strain evaluation; Microfermentation; Phosphate; Protein expression.
© 2024 The Authors.