Cell-free systems provide a versatile platform for the development of low-cost, easy-to-use sensors diverse analytes. However, sensor affinity dictates response sensitivity, and improving binding can be challenging. Here, we describe efforts to address this problem while developing biosensor vitamin B12, critical micronutrient. We first use B12-responsive transcription factor enable B12-dependent output in cell-free reaction, but resulting responds B12 far above clinically relevant...

The field of metabolic engineering has yielded remarkable accomplishments in using cells to produce valuable molecules, and cell-free expression (CFE) systems have the potential push even further. However, CFE still face some outstanding challenges, including endogenous activity that is poorly understood yet a significant impact on productivity. Here, we use metabolomics characterize temporal changes their constituent components, central carbon amino acid metabolism. We find while changing...

Hyperhomocysteinemia─a condition characterized by elevated levels of homocysteine in the blood─is associated with multiple health conditions including folate deficiency and birth defects, but there are no convenient, low-cost methods to measure plasma. A cell-free biosensor that harnesses native sensing machinery Escherichia coli bacteria could satisfy need for a detection platform these characteristics. Here, we describe our efforts engineer point-of-care, assessment status. This can detect...

Cell lysis─by sonication or bead beating, for example─is a key step in preparing extracts cell-free expression systems. To create high protein-production capacity extracts, standard practice is to lyse cells sufficiently thoroughly disrupt the membrane and thus extract machinery but without degrading that machinery. Here, we investigate impact of different energy inputs on Escherichia coli extracts. While existence operator-specific optimal widely known, our findings show input yields...

Although cell-free protein expression has been widely used for the synthesis of single proteins, synthetic biology rapidly expanded to new, more complex applications. One such application is prototyping or implementation genetic networks involving multiple proteins at precise ratios, often from different plasmids. However, plasmids may inadvertently result in unexpected, off-target changes levels being expressed, a phenomenon termed plasmid crosstalk. Here, we show that effects...

Vitamin C (l-ascorbate) deficiency is a global public health issue most prevalent in resource-limited regions, creating need for an inexpensive detection platform. Here, we describe efforts to engineer whole-cell and cell-free ascorbate biosensors. Both sensors used the protein UlaR, which binds metabolite of regulates transcription. The sensor could detect lower, physiologically relevant concentrations ascorbate, attributed intact functionality phosphotransferase system (PTS) that...

Abstract The field of metabolic engineering has yielded remarkable accomplishments in using cells to produce valuable molecules, and cell-free expression (CFE) systems have the potential push even further. However, CFE still face some outstanding challenges, including endogenous activity that is poorly understood yet a significant impact on productivity. Here, we use metabolomics characterize temporal changes their constituent components, central carbon amino acid metabolism. We find while...