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Aqueous two-phase systems (ATPSs) have long been used for the facile and rapid extraction of biomolecules of interest. Selective partitioning of DNA is useful for nucleic acid purification and in the design of novel sensing technologies. This paper investigates the partitioning of a plasmid within a poorly understood ATPS comprising the polymers poly(ethylene glycol) (PEG) 35 kDa and Ficoll 400 kDa. The focus is placed on dissecting the compositional effects of the ATPS—that is, whether set concentrations of physiological ions or the polymers themselves can tune DNA phase preference and strength of partitioning. The work here uncovers the antagonistic effects of magnesium and ammonium ions, as well as the role that phase-forming polymer partitioning plays in plasmid enrichment. Testing the ions in conjunction with different ATPS formulations highlights the complexity of the system at hand, prompting the exploration of DNA’s conformational changes in response to polymer and salt presence. The work presented here offers multiple optimization parameters for downstream applications of PEG–Ficoll ATPSs, such as in vitro transcription/translation-based biosensing, in which performance is heavily dependent upon nucleic acid partitioning. 

Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.