Co-culture techniques find myriad applications in biology for studying natural or synthetic interactions between cell populations. Such techniques are of great importance in synthetic biology, as multi-species cell consortia and other natural or synthetic ecology systems are widely seen to hold enormous potential for foundational research as well as novel industrial, medical and environmental applications with many proof-of-principle studies in recent years. What is needed for co-cultures to fulfil their potential? Cell–cell interactions in co-cultures are strongly influenced by the extracellular environment, which is determined by the experimental set-up, which therefore needs to be given careful consideration. An overview of existing experimental and theoretical co-culture set-ups in synthetic biology and adjacent fields is given here, and challenges and opportunities involved in such experiments are discussed. Greater focus on foundational technology developments for co-cultures is needed for many synthetic biology systems to realize their potential in both applications and answering biological questions.

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterize in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in E. coli synthetic biology.

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. Significance Nonmodel bacteria have essential roles to play in the future development of biotechnology by providing new sources of biocatalysts, antibiotics, hosts for bioproduction, and engineered “living therapies.” The characterization of such hosts can be challenging, as many are not tractable to standard molecular biology techniques. This paper presents a rapid and automated methodology for characterizing new DNA parts from a nonmodel bacterium using cell-free transcription–translation. Data analysis was performed with Bayesian parameter inference to provide an understanding of gene-expression dynamics and resource sharing. We suggest that our integrated approach is expandable to a whole range of nonmodel bacteria for the characterization of new DNA parts within a native cell-free background for new biotechnology applications.

Advancing synthetic biology to the multicellular level requires the development of multiple cell-to-cell communication channels that propagate information with minimal signal interference. The development of quorum-sensing devices, the cornerstone technology for building microbial communities with coordinated system behaviour, has largely focused on cognate acyl-homoserine lactone (AHL)/transcription factor pairs, while the use of non-cognate pairs as a design feature has received limited attention. Here, we demonstrate a large library of AHL-receiver devices, with all cognate and non-cognate chemical signal interactions quantified, and we develop a software tool that automatically selects orthogonal communication channels. We use this approach to identify up to four orthogonal channels in silico, and experimentally demonstrate the simultaneous use of three channels in co-culture. The development of multiple non-interfering cell-to-cell communication channels is an enabling step that facilitates the design of synthetic consortia for applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation and spatial organisation.

The success of Pichia pastoris as a heterologous expression system lies predominantly in the impressive yields that can be achieved due to high volumetric productivity. However, low specific productivity still inhibits the potential success of this platform. Multi-(gene) copy clones are potentially a quick and convenient method to increase recombinant protein titer, yet they are not without their pitfalls. It has been more than twenty years since the first reported use of multi-copy clones and it is still an active area of research to find the fastest and most efficient method for generating these strains. It has also become apparent that there is not always a linear correlation between copy number and protein titer, leading to in-depth investigations into how to minimize the negative impact of secretory stress and achieve clonal stability.