The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations

Abstract

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 optimal protocols used 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, the development approach described is adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.

Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

“…Microfermentations were conducted in 96-well plates (Genesee Scientific, San Diego, CA, Cat#25-104) using a previously reported method 43,46,86 . Glycerol stocks of each strain were used to inoculate LB overnight cultures in the 96-well plates, with 2.5μL of glycerol stock in 100μL of LB media per well.…”

“…Lastly, we also evaluated the scalability of VHH production with the autoDC REdox strain, ranging from small-scale microfermentations to a 1L bioreactor 43,86 . We confirmed high-titer expression in 96-well plates (Figure 5B and S10) by leveraging autolysis enzymes for efficient lysis, a crucial component of our engineered system that overcomes potential challenges in the absence of this mechanism 58,[87][88][89] .…”