Sensbio: an online server for biosensor design.

Allosteric transcription factor (aTF) based biosensors can be used to engineer genetic circuits for a wide range of applications. The literature and online databases contain hundreds of experimentally validated molecule-TF pairs; however, the knowledge is scattered and often incomplete. Additionally, compared to the number of compounds that can be produced in living systems, those with known associated TF-compound interactions are low. For these reasons, new tools that help researchers find new possible TF-ligand pairs are called for. In this work, we present Sensbio, a computational tool that through similarity comparison against a TF-ligand reference database, is able to identify putative transcription factors that can be activated by a given input molecule. In addition to the collection of algorithms, an online application has also been developed, together with a predictive model created to find new possible matches based on machine learning.

Transcription factor-based biosensors for screening and dynamic regulation.

Advances in synthetic biology and genetic engineering are bringing into the spotlight a wide range of bio-based applications that demand better sensing and control of biological behaviours. Transcription factor (TF)-based biosensors are promising tools that can be used to detect several types of chemical compounds and elicit a response according to the desired application. However, the wider use of this type of device is still hindered by several challenges, which can be addressed by increasing the current metabolite-activated transcription factor knowledge base, developing better methods to identify new transcription factors, and improving the overall workflow for the design of novel biosensor circuits. These improvements are particularly important in the bioproduction field, where researchers need better biosensor-based approaches for screening production-strains and precise dynamic regulation strategies. In this work, we summarize what is currently known about transcription factor-based biosensors, discuss recent experimental and computational approaches targeted at their modification and improvement, and suggest possible future research directions based on two applications: bioproduction screening and dynamic regulation of genetic circuits.

Versioning biological cells for trustworthy cell engineering.

"Full-stack" biotechnology platforms for cell line (re)programming are on the horizon, thanks mostly to (a) advances in gene synthesis and editing techniques as well as (b) the growing integration of life science research with informatics, the internet of things and automation. These emerging platforms will accelerate the production and consumption of biological products. Hence, traceability, transparency, and-ultimately-trustworthiness is required from cradle to grave for engineered cell lines and their engineering processes. Here we report a cloud-based version control system for biotechnology that (a) keeps track and organizes the digital data produced during cell engineering and (b) molecularly links that data to the associated living samples. Barcoding protocols, based on standard genetic engineering methods, to molecularly link to the cloud-based version control system six species, including gram-negative and gram-positive bacteria as well as eukaryote cells, are shown. We argue that version control for cell engineering marks a significant step toward more open, reproducible, easier to trace and share, and more trustworthy engineering biology.

Fast biofoundries: coping with the challenges of biomanufacturing.

Biofoundries are highly automated facilities that enable the rapid and efficient design, build, test, and learn cycle of biomanufacturing and engineering biology, which is applicable to both research and industrial production. However, developing a biofoundry platform can be expensive and time consuming. A biofoundry should grow organically, starting from a basic platform but with a vision for automation, equipment interoperability, and efficiency. By thinking about strategies early in the process through process planning, simulation, and optimization, bottlenecks can be identified and resolved. Here, we provide a survey of technological solutions in biofoundries and their advantages and limitations. We explore possible pathways towards the creation of a functional, early-phase biofoundry, and strategies towards long-term sustainability.

Targetron-Assisted Delivery of Exogenous DNA Sequences into Pseudomonas putida through CRISPR-Aided Counterselection.

Genome editing methods based on group II introns (known as targetron technology) have long been used as a gene knockout strategy in a wide range of organisms, in a fashion independent of homologous recombination. Yet, their utility as delivery systems has typically been suboptimal due to the reduced efficiency of insertion when carrying exogenous sequences. We show that this limitation can be tackled and targetrons can be adapted as a general tool in Gram-negative bacteria. To this end, a set of broad-host-range standardized vectors were designed for the conditional expression of the Ll.LtrB intron. After establishing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths, to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. With these novel tools, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its ΔrecA derivative. Finally, we applied this technology to successfully tag P. putida with an orthogonal short sequence barcode that acts as a unique identifier for tracking this microorganism in biotechnological settings. These results show the value of the targetron approach for the unrestricted delivery of small DNA fragments to precise locations in the genomes of Gram-negative bacteria, which will be useful for a suite of genome editing endeavors.

Prototyping of microbial chassis for the biomanufacturing of high-value chemical targets.

Metabolic engineering technologies have been employed with increasing success over the last three decades for the engineering and optimization of industrial host strains to competitively produce high-value chemical targets. To this end, continued reductions in the time taken from concept, to development, to scale-up are essential. Design-Build-Test-Learn pipelines that are able to rapidly deliver diverse chemical targets through iterative optimization of microbial production strains have been established. Biofoundries are employing in silico tools for the design of genetic parts, alongside combinatorial design of experiments approaches to optimize selection from within the potential design space of biological circuits based on multi-criteria objectives. These genetic constructs can then be built and tested through automated laboratory workflows, with performance data analysed in the learn phase to inform further design. Successful examples of rapid prototyping processes for microbially produced compounds reveal the potential role of biofoundries in leading the sustainable production of next-generation bio-based chemicals.

Protein interactions within and between two F-type type IV secretion systems.

Bacterial type IV secretion systems (T4SSs) can mediate conjugation. The T4SS from Neisseria gonorrhoeae possesses the unique ability to mediate DNA secretion into the extracellular environment. The N. gonorrhoeae T4SS can be grouped with F-type conjugative T4SSs based on homology. We tested 17 proteins important for DNA secretion by N. gonorrhoeae for protein interactions. The BACTH-TM bacterial two-hybrid system was successfully used to study periplasmic interactions. By determining if the same interactions were observed for F-plasmid T4SS proteins and when one interaction partner was replaced by the corresponding protein from the other T4SS, we aimed to identify features associated with the unique function of the N. gonorrhoeae T4SS as well as generic features of F-type T4SSs. For both systems, we observed already described interactions shared by homologs from other T4SSs as well as new and described interactions between F-type T4SS-specific proteins. Furthermore, we demonstrate, for the first-time, interactions between proteins with homology to the conserved T4SS outer membrane core proteins and F-type-specific proteins and we confirmed two of them by co-purification. The F-type-specific protein TraHN was found to localize to the outer membrane and the presence of significant amounts of TraHN in the outer membrane requires TraGN .

Linking Engineered Cells to Their Digital Twins: A Version Control System for Strain Engineering.

As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biological engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnology and digital technology, a deficit in software and laboratory techniques diminishes the ability to make biotechnology more agile, reproducible, and transparent while, at the same time, limiting the security and safety of synthetic biology constructs. To partially address some of these problems, this paper presents an approach for physically linking engineered cells to their digital footprint-we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialized version control system for collaborative strain engineering via simple barcoding protocols.