21EngBio: Engineering Bioprogrammable Materials Using Hydrogel-Based Cell-Free Gene Expression and Spatiotemporal Modelling

Living systems possess many and varied molecular and biochemical means to detect and respond to a wide range of internal and external stimuli. This includes identifying competitors, predators or prey, and the identification of sources of food or chemicals that may cause harm. At a cellular level, these stimuli are frequently detected through the binding of a chemical with a protein partner. This may directly alter gene expression or may trigger further signalling pathways within the cell. Using these mechanisms, cells continually monitor multiple inputs leading to a range of outcomes. These may be biochemical changes (for example, producing enzymes to detoxify substances or exploit new sources of food) or they may be changes that permit growth towards more favourable conditions. From a developmental perspective, the integration of internal and external signalling is crucial for the development of an organism's body plan. What appear as complex responses however, frequently emerge from simple, local molecular interactions coupled with the diffusion of signal molecules. Being able to replicate some of these cellular aspects of living systems in materials - without using living cells - could be transformative to a range of applications. This may permit the development of materials capable of detecting specific pathogens and the subsequent production of targeted antimicrobials, materials that alert the user to the presence of noxious chemicals, or even self-organisation of multiple functions at a molecular level. The diffusion of a small number of chemical signals and their impact on gene expression for example, may explain the placement of taste receptors on the tongue and the development of camouflage patterns. Using living systems within materials however, is problematic. It requires the maintenance of a benign environment and an understanding of existing cellular regulatory mechanisms, while the release of genetically modified organisms is not desirable in many regions.

Our goal is to use cellular molecular and biochemical networks in materials free from the constraints of living cells. We have recently demonstrated cell-free gene expression inside hydrogels with different physical properties and end-user applications and we have used cell-free protein synthesis reactions to alter the properties of the materials themselves. Further, the use of molecular biology and biochemical components outside of a cell allows integration with non-living chemistries that is not possible to enact within a living organism. For example, we are currently combining enzymatic activities with cell-free gene expression to create materials for passive surveillance of viruses and vectors of viruses (BB/V017209/1, BB/V017551/1).

Here we will generate the foundational experimental data and mathematical models that describe the relationship between diffusion and gene expression in three dimensional hydrogel matrices. We will use this understanding to design gene networks that can be homogeneously distributed but result in heterogeneous functionality. In addition, we will use this opportunity to examine some of the broader considerations around the concept of bioprogrammable matter, and use this dialogue to inform future research programmes.

Our goal is to embed cell-free molecular and biochemical processes for detection, computation and response into hydrogel materials. Reliable engineering of hydrogel-based cell-free systems requires a modelling approach that captures spatial as well as temporal behaviour. These models need to be supported by a knowledge of the diffusion coefficients for given biomolecules in specific matrices. In particular quantification of the movement of transcription factors through hydrogel matrices, alongside an understanding of how this affects gene expression and the transmission of genetically encoded signals. In brief, modelling synthetic gene networks in three dimensional structures must take account of the impact that spatial organisation plays in regulating gene expression.

This project comprises four objectives. Objective 1 directly measures diffusion of fluorescently labelled transcription factors in polymer matrices, examining how changes in polymer concentration and transcription factors affect diffusion rates, and the impact that diffusion of transcription factors has on gene expression distal to the site of activation. Objective 2 develops spatiotemporal models that describe gene expression in these experimental systems, refined and validated against data collected in O1. Objective 3 will use this understanding to help forward engineer two homogeneously distributed gene networks with different goals. The first will respond to a stimuli but reset to its original state when the stimuli is removed. The second will remember stimulation after its removal but only locally. For both of these goals the data and models developed in O1 and 2 will be required to guide establishment of appropriate diffusion gradients. Finally, Objective 4 employs the AREA Responsible Research and Innovation framework to consider the broad implications of bioprogrammable matter such that it may inform future research programmes.