Genetically-encoded biosensors for monitoring cellular stress in bioprocessing Karen M Polizzi 1,2 and Cleo Kontoravdi 3 With the current wealth of transcriptomic data, it is possible to design genetically-encoded biosensors for the detection of stress responses and apply these to high-throughput bioprocess development and monitoring of cellular health. Such biosensors can sense extrinsic factors such as nutrient or oxygen deprivation and shear stress, as well as intrinsic stress factors like oxidative damage and unfolded protein accumulation. Alongside, there have been developments in biosensing hardware and software applicable to the field of genetically-encoded biosensors in the near future. This review discusses the current state-of-the-art in biosensors for monitoring cultures during biological manufacturing and the future challenges for the field. Connecting the individual achievements into a coherent whole will enable the application of genetically-encoded biosensors in industry. Addresses 1 Department of Life Sciences, Imperial College London, London SW7 2AZ, UK 2 Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK 3 Department of Chemical Engineering and Chemical Technology, Imperial College London, London SW7 2AZ, UK Corresponding author: Polizzi, Karen M (k.polizzi@imperial.ac.uk) Current Opinion in Biotechnology 2015, 31:50–56 This review comes from a themed issue on Analytical biotechnology Edited by Hadley D Sikes and Nicola Zamboni http://dx.doi.org/10.1016/j.copbio.2014.07.011 0958-1669/# 2014 Elsevier Ltd. All right reserved. Introduction The efficient development of industrial processes for manufacturing biological products remains a challenge. Ensuring batch-to-batch reproducibility and maximising the yield from production vessels requires both a sys- tematic investigation of the best operating parameters during process development and analysis of cultures during production to ensure quality. High-throughput culture systems such as the Micro-24 MicroReactor System (Pall Life Sciences), the Ambr TM workstation (TAP Biosystems) and the BioLector 1 (m2p- labs) can accelerate process development. These systems allow monitoring of, and in some cases control over, temperature, dissolved oxygen and pH. The low culture volume, however, makes it difficult to obtain samples for other analyses. In situ sensing of analytes of interest is therefore an attractive option for real-time high-through- put data generation from such platforms, because it removes the need for sampling. The same sensors can then be used to monitor cultures during production to confirm that the analytes remain within the limits necess- ary to maintain productivity and take corrective action when they do not. There are several studies outlining recent developments in biosensor design and application to bioprocessing, including biosensors to directly sense key nutrients, such as sugars and amino acids (e.g. glucose and glutamine biosensors [1], and other efforts recently reviewed in [2  ]). In addition to metabolism, effectors of the unfolded protein response have been shown to positively correlate with specific productivity of recombinant proteins [3–5], while oxidative damage from metabolism and/or protein misfolding can accumulate over time, leading to redox imbalance and eventually cell death [6,7]. Extrinsic pro- cess parameters such as shear stress, nutrient and oxygen depletion can also affect performance by activating cel- lular stress responses. This review focuses on the use of genetically-encoded biosensors to sense extrinsic and intrinsic cellular stress, which can indicate the need to change operating parameters in order to improve cell health and productivity. Biosensors can also be used to establish the limits of feasible operating parameters and for high throughput design of media. We have focused on biosensors that rely on the activation of transcription as their output, as these can be developed from the wealth of information from recent transcriptomic studies of stress responses (e.g. [8,9,10  ,11]) in a relatively modular fashion (Figure 1). Genetically-encoded biosensors are pieces of DNA that contain the instructions for a biosensor circuit. The output of the circuit is usually a reporter protein that is easy to detect. The classical example is the green fluor- escent protein (GFP) and its derivatives. Others include enzymes that catalyse a reaction that produces a colour change (alkaline phosphatase, beta-galactosidase, etc.) and luciferase, which utilises the substrate luciferin, ATP, and oxygen to produce a detectable light signal. In order to monitor dynamic variations, reporter proteins can be destabilised through the addition of degradation tags ([12–14]). Available online at www.sciencedirect.com ScienceDirect Current Opinion in Biotechnology 2015, 31:50–56 www.sciencedirect.com