Microbial cell factories offer an attractive approach for production of biobased products.Unfortunately, designing, building, and optimizing biosynthetic pathways remains a complex challenge, especially for industrially-relevant, non-model organisms. To address this challenge, we describe a platform for in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (iPROBE). In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE with two examples. First, we tested and ranked 54 different pathways for 3-hydroxybutyrate production, improving in vivo production in Clostridium by 20-fold to 14.63 ± 0.48 g/L and identifying a new biosynthetic route to (S)-(+)-1,3butanediol. Second, we used iPROBE and data-driven design to optimize a 6-step n-butanol pathway, increasing titers 4-fold across 205 pathways, and showed strong correlation between cell-free and cellular performance. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

"Non-fouling" polymer brush surfaces can greatly improve the performance of in vitro diagnostic (IVD) assays due to the reduction of non-specific protein adsorption and consequent improvement of signal-to-noise ratios. Herein, the development of synthetic polymer brush architectures that suppress adventitious protein adsorption is reviewed and their integration into surface plasmon resonance and fluorescent sandwich immunoassay formats is discussed. Also, highlighted is a novel, self-contained immunoassay platform (the D4 assay), that transforms time-consuming laboratory-based assays into a user-friendly and point-of-care format with a sensitivity and specificity comparable or better than standard ELISA directly from unprocessed samples. These advancements clearly demonstrate the This article is protected by copyright. All rights reserved. 2 utility of non-fouling polymer brushes as a substrate for ultra-sensitive and robust diagnostic assays that may be suitable for clinical testing, in field and laboratory settings.

Poly(ethylene glycol) (PEG), a linear polymer known for its “stealth” properties, is commonly used to passivate the surface of biomedical implants and devices, and it is conjugated to biologic drugs to improve their pharmacokinetics. However, its antigenicity is a growing concern. Here, the antigenicity of PEG is investigated when assembled in a poly(oligoethylene glycol) methacrylate (POEGMA) “bottlebrush” configuration on a planar surface. Using ethylene glycol (EG) repeat lengths of the POEGMA sidechains as a tunable parameter for optimization, POEGMA brushes with sidechain lengths of two and three EG repeats are identified as the optimal polymer architecture to minimize binding of anti‐PEG antibodies (APAs), while retaining resistance to nonspecific binding by bovine serum albumin and cultured cells. Binding of backbone‐ versus endgroup‐selective APAs to POEGMA brushes is further investigated, and finally the antigenicity of POEGMA coatings is assessed against APA‐positive clinical plasma samples. These results are applied toward fabricating immunoassays on POEGMA surfaces with minimal reactivity toward APAs while retaining a low limit‐of‐detection for the analyte. Taken together, these results offer useful design concepts to reduce the antigenicity of polymer brush‐based surface coatings used in applications involving human or animal matrices.