The new generation of cell-free gene expression systems enables prototyping and engineering biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription-translation (TXTL) platforms remains a major goal to either execute larger DNA programs, or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch mode reactions, synthesis of the fluorescent reporter protein eGFP reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced to concentrations of more than 8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kbp and about 60 genes, is produced at a concentration of 1013 PFU/ml. This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for either testing regulatory elements and circuits, biomanufacturing biologics, or building synthetic cells.

This study clarifies, in broad terms, how biology majors differ from those not intending to major in STEM fields. The article concludes with suggestions for inclusive teaching for future scientists as well as an informed citizenry.

Elemental homeostasis has been largely characterized using three important elements that were part of the Redfield ratio (i.e., carbon: nitrogen: phosphorus). These efforts have revealed substantial diversity in homeostasis among taxonomic groups and even within populations. Understanding the evolutionary basis, and ecological consequences of such diversity is a central challenge. Here, we propose that a more complete understanding of homeostasis necessitates the consideration of other elements beyond C, N, and P. Specifically, we posit that physiological complexity underlying maintenance of elemental homeostasis along a single elemental axis impacts processing of other elements, thus altering elemental homeostasis along other axes. Indeed, transcriptomic studies in a wide variety of organisms have found that individuals differentially express significant proportions of the genome in response to variability in supply stoichiometry in order to maintain varying levels of homeostasis. We review the literature from the emergent field of ionomics that has established the consequences of such physiological trade-offs on the content of the entire suite of elements in an individual. Further, we present experimental data on bacteria exhibiting divergent phosphorus homeostasis phenotypes demonstrating the fundamental interconnectedness among elemental quotas. These observations suggest that physiological adjustments can lead to unexpected patterns in biomass stoichiometry, such as correlated changes among suites of non-limiting microelements in response to limitation by macroelements. Including the entire suite of elements that comprise biomass will foster improved quantitative understanding of the links between chemical cycles and the physiology of organisms.

Course-based undergraduate research experiences (CUREs) are a type of laboratory learning environment associated with a science course, in which undergraduates participate in novel research. According to Auchincloss et al. (CBE Life Sci Educ 2104; 13:29–40), CUREs are distinct from other laboratory learning environments because they possess five core design components, and while national calls to improve STEM education have led to an increase in CURE programs nationally, less work has specifically focused on which core components are critical to achieving desired student outcomes. Here we use a backward elimination experimental design to test the importance of two CURE components for a population of non-biology majors: the experience of discovery and the production of data broadly relevant to the scientific or local community. We found nonsignificant impacts of either laboratory component on students’ academic performance, science self-efficacy, sense of project ownership, and perceived value of the laboratory experience. Our results challenge the assumption that all core components of CUREs are essential to achieve positive student outcomes when applied at scale.