Characterizing chemical signaling between engineered “microbial sentinels” in porous microplates

Abstract

Living materials combine a material scaffold, that is often porous, with engineered cells that perform sensing, computing, and biosynthetic tasks. Designing such systems is difficult because little is known regarding signaling transport parameters in the material. Here, the development of a porous microplate is presented. Hydrogel barriers between wells have a porosity of 60% and a tortuosity factor of 1.6, allowing molecular diffusion between wells. The permeability of dyes, antibiotics, inducers, and quorum signals between wells were characterized. A “sentinel” strain was constructed by introducing orthogonal sensors into the genome of Escherichia coli MG1655 for IPTG, anhydrotetracycline, L‐arabinose, and four quorum signals. The strain’s response to inducer diffusion through the wells was quantified up to 14 mm, and quorum and antibacterial signaling were measured over 16 h. Signaling distance is dictated by hydrogel adsorption, quantified using a linear finite element model that yields adsorption coefficients from 0 to 0.1 mol m−3. Parameters derived herein will aid the design of living materials for pathogen remediation, computation, and self‐organizing biofilms.

Synthetic Homoserine Lactone Sensors for Gram-Positive Bacillus subtilis Using LuxR-Type Regulators

“…53,54 However, we also observed crosstalk in B. subtilis for 3OC6-HSL with CinR, which was not observed in E. coli. 54,106 The sensor output and resulting crosstalk for a quorum sensor can be affected by many factors, including the concentration of the molecular signals and regulator expression, and overexpression of the regulator has been shown to increase promiscuity. 107−109 Decreasing the expression of CinR may mitigate this observed signal crosstalk; yet, it could be due to other differences of B. subtilis.…”

A roadmap to establish a comprehensive platform for sustainable manufacturing of natural products in yeast

“…Alternatively, living materials combining a material scaffold with a porous structure may address the imbalanced co-culture composition issue by enabling the spatial organization of SMC species in cocultures as that in nature (where spatial organization enables multiple species to coexist) 60 . Recently a methacrylate HEMA-EDMA porous microplate living material has been designed to maximize neighboring well interactions 61 . This culturing system enables exchange of small molecules, but not cells, between wells 61 .…”

“…Recently a methacrylate HEMA-EDMA porous microplate living material has been designed to maximize neighboring well interactions 61 . This culturing system enables exchange of small molecules, but not cells, between wells 61 . With this porous microplate, a spatially segregated co-culture of autotrophic bacteria and yeast can potentially be established.…”

“…With this porous microplate, a spatially segregated co-culture of autotrophic bacteria and yeast can potentially be established. However, extensive follow-up work must be performed on the co-culture of autotrophic bacteria and yeast 61 . Another technique called microbial swarmbot consortia (MSBC) has been created by encapsulating subpopulations in polymeric microcapsules 62 .…”

Identifying LasR Quorum Sensors with Improved Signal Specificity by Mapping the Sequence–Function Landscape

“…Various methods have been employed to alter ligand specificity of allosteric transcription factors. Directed evolution via random mutagenesis (by error-prone PCR or gene shuffling) of the ligand binding domain or protein coding sequence has been widely used to engineer ligand specificity, including for improved molecular discrimination and for preferential specificity for molecules other than the natural cognate ligand. − While random mutagenesis can introduce numerous mutations and generate sequence diversity, it often suffers from mutational bias that hinders screening all amino acid substitutions and therefore cannot comprehensively determine sequence–function relationships. − Another approach is designing transcription factors using rational design that applies structure-based computational modeling and has also been used to achieve desired ligand specificity, − yet it requires a large amount of prior knowledge and predictions of biophysical interactions that are often not known . Other studies have chosen the middle ground of semirational design, which utilizes available structural and functional information on the transcription factor to choose the target residues and amino acid diversity for protein engineering. − Semirational design can narrow the designed sequence space to a subset within the limits of what can be experimentally tested and predicted to have a greater likelihood for a desired functionality …”