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Cell-free protein synthesis (CFPS) is an established biotechnology tool that has shown great utility in many applications such as prototyping proteins, building genetic circuits, designing biosensors, and expressing cytotoxic proteins. Although CFPS has been widely deployed, the many, varied methods presented in the literature can be challenging for new users to adopt. From our experience and others who newly enter the field, one of the most frustrating aspects of applying CFPS as a laboratory can be the large levels of variability that are present within experimental replicates. Herein we provide a retrospective summary of CFPS methods that reduce variability significantly. These methods include optimized extract preparation, fully solubilizing the master mix components, and careful mixing of the reaction. These have reduced our coefficient of variation from 97.3% to 1.2%. Moreover, these methods allow complete novices (e.g. semester rotation undergraduate students) to provide data that is comparable to experienced users, thus allowing broader participation in this exciting research area.

In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell‐based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost‐optimized, scalably‐manufactured Escherichia coli extract and minimal supplement reagents to perform cell‐free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti‐GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1–7)M(2–9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.