Towards the High–Throughput Construction of Fluorescent Biosensors

Rapid quantification of in vivo metabolite concentrations is a significant challenge in understanding and engineering metabolism. It has been demonstrated that single fluorescent protein biosensors can reliably act as in vivo sensors of metabolite concentrations with high specificity, a linear response, a wide dynamic range and on relevant spatiotemporal scales. However, rational approaches to the construction of such sensors are time consuming and difficult, resulting in few reported examples. Therefore, the high-throughput construction of fluorescent protein-based biosensors would greatly enhance our abilities to meet the current and future challenges of probing and manipulating metabolism. Towards this, we report on the development of a general, transposon-mediated strategy that was applied to the construction of a sensor for trehalose, an important metabolite for which a reliable sensor is lacking. In this approach, a modified transposon was used to create a library of green fluorescent protein insertions within T. litoralis D-trehalose/D-maltose-binding protein (TMBP). The library was then subjected to successive rounds of fluorescence activated cell sorting (FACS) in the presence and absence of trehalose to obtain a sensor with high sensitivity and specificity. From these results, we have learned about the generality of this technique for constructing fluorescent biosensors.