A bacteriocin expression platform for targeting pathogenic bacterial species.

Bacteriocins are antimicrobial peptides that are naturally produced by many bacteria. They hold great potential in the fight against antibiotic resistant bacteria, including ESKAPE pathogens. Engineered live biotherapeutic products (eLBPs) that secrete bacteriocins can be created to deliver targeted bacteriocin production. Here we develop a modular bacteriocin secretion platform that can be used to express and secrete multiple bacteriocins from non-pathogenic Escherichia coli host strains. As a proof of concept we create Enterocin A (EntA) and Enterocin B (EntB) secreting strains that show strong antimicrobial activity against Enterococcus faecalis and Enterococcus faecium in vitro, and characterise this activity in both solid culture and liquid co-culture. We then develop a Lotka-Volterra model that can be used to capture the interactions of these competitor strains. We show that simultaneous exposure to EntA and EntB can delay Enterococcus growth. Our system has the potential to be used as an eLBP to secrete additional bacteriocins for the targeted killing of pathogenic bacteria.

Emergent digital bio-computation through spatial diffusion and engineered bacteria.

Biological computing is a promising field with potential applications in biosafety, environmental monitoring, and personalized medicine. Here we present work on the design of bacterial computers using spatial patterning to process information in the form of diffusible morphogen-like signals. We demonstrate, mathematically and experimentally, that single, modular, colonies can perform simple digital logic, and that complex functions can be built by combining multiple colonies, removing the need for further genetic engineering. We extend our experimental system to incorporate sender colonies as morphogen sources, demonstrating how one might integrate different biochemical inputs. Our approach will open up ways to perform biological computation, with applications in bioengineering, biomaterials and biosensing. Ultimately, these computational bacterial communities will help us explore information processing in natural biological systems.

Microbiome engineering: engineered live biotherapeutic products for treating human disease.

The human microbiota is implicated in many disease states, including neurological disorders, cancer, and inflammatory diseases. This potentially huge impact on human health has prompted the development of microbiome engineering methods, which attempt to adapt the composition and function of the human host-microbiota system for a therapeutic purpose. One promising method is the use of engineered microorganisms that have been modified to perform a therapeutic function. The majority of these products have only been demonstrated in laboratory models; however, in recent years more concepts have reached the translational stage. This has led to an increase in the number of clinical trials, which are designed to assess the safety and efficacy of these treatments in humans. Within this review, we highlight the progress of some of these microbiome engineering clinical studies, with a focus on engineered live biotherapeutic products.

Engineered acetoacetate-inducible whole-cell biosensors based on the AtoSC two-component system.

Whole-cell biosensors hold potential in a variety of industrial, medical, and environmental applications. These biosensors can be constructed through the repurposing of bacterial sensing mechanisms, including the common two-component system (TCS). Here we report on the construction of a range of novel biosensors that are sensitive to acetoacetate, a molecule that plays a number of roles in human health and biology. These biosensors are based on the AtoSC TCS. An ordinary differential equation model to describe the action of the AtoSC TCS was developed and sensitivity analysis of this model used to help inform biosensor design. The final collection of biosensors constructed displayed a range of switching behaviours at physiologically relevant acetoacetate concentrations and can operate in several Escherichia coli host strains. It is envisaged that these biosensor strains will offer an alternative to currently available commercial strip tests and, in future, may be adopted for more complex in vivo or industrial monitoring applications.

Detecting Changes in the Caenorhabditis elegans Intestinal Environment Using an Engineered Bacterial Biosensor.

Caenorhabditis elegans has become a key model organism within biology. In particular, the transparent gut, rapid growing time, and ability to create a defined gut microbiota make it an ideal candidate organism for understanding and engineering the host microbiota. Here we present the development of an experimental model that can be used to characterize whole-cell bacterial biosensors in vivo. A dual-plasmid sensor system responding to isopropyl ß-d-1-thiogalactopyranoside was developed and fully characterized in vitro. Subsequently, we show that the sensor was capable of detecting and reporting on changes in the intestinal environment of C. elegans after introducing an exogenous inducer into the environment. The protocols presented here may be used to aid the rational design of engineered bacterial circuits, primarily for diagnostic applications. In addition, the model system may serve to reduce the use of current animal models and aid in the exploration of complex questions within general nematode and host-microbe biology.