YeastFab is a Golden Gate-based cloning standard and parts repository. It is designed for modular, hierarchical assembly of transcription units and multi-gene assemblies for expression in Saccharomyces cerevisiae. This makes it a suitable toolbox to optimize the expression strength of heterologous genes in yeast. When cloning heterologous coding sequences into YeastFab vectors, in several cases we have observed toxicity to the cloning host Escherichia coli. The provided protocol details how to clone such toxic genes from multiple synthetic DNA fragments while adhering to the YeastFab standard. The presented cloning strategy includes a C-terminal FLAG tag that allows screening for constructs with a desired protein expression in yeast by western blot. The design allows scarlessly removing the tag through a Golden Gate reaction to facilitate cloning of expression constructs with the native, untagged transgene.

Devices sensing inputs and generating outputs are fundamental regulatory units, and as such are the basis of more complex regulatory networks. We provide an overview of regulatory devices used as fundamental regulatory building blocks in synthetic biology, and how complex genetic circuitry is being constructed from them. We first comprehensively explore devices operating at different levels of gene regulation, with action modes on the DNA sequence, to transcriptional, translational and post-translational control. We then discuss design principles of constructing genetic circuits from basic regulatory units, addressing challenges such as orthogonality, context-dependence, noise, and complexity. We present examples of genetic circuitry, including bistable switches, logic gates, signal amplification, memory devices and circuitry for biocomputation. How artificial genetic circuitry can be useful in real-life applications is illustrated with examples from bioproduction, living therapeutics, and biosafety. Our aim is to provide a comprehensive overview of the toolbox of regulatory devices and a profound understanding of their potential for constructing diverse genetic circuits and their applications.

The dual genetic control of mitochondrial respiratory function, combined with the high mutation rate of the mitochondrial genome (mtDNA), makes mitochondrial diseases among the most frequent genetic diseases in humans (1 in 5,000 in adults). With no effective treatments available, gene therapy approaches have been proposed. Notably, several studies have demonstrated the potential for nuclear expression of a healthy copy of a dysfunctional mitochondrial gene, referred to as allotopic expression, to help recover respiratory function. However, allotopic expression conditions require significant optimization. We harnessed engineering biology tools to improve the allotopic expression of the COX2-W56R gene in the budding yeast Saccharomyces cerevisiae. Through conducting random mutagenesis and screening of the impact of vector copy number, promoter, and mitochondrial targeting sequence, we substantially increased the mitochondrial incorporation of the allotopic protein and significantly increased recovery of mitochondrial respiration. Moreover, CN-PAGE analyses revealed that our optimized allotopic protein does not impact cytochrome c oxidase assembly, or the biogenesis of respiratory chain supercomplexes. Importantly, the most beneficial amino acid substitutions found in the second transmembrane helix (L93S and I102K) are conserved residues in the corresponding positions of human MT-CO2 (L73 and L75), and we propose that mirroring these changes could potentially help improve allotopic Cox2p expression in human cells. To conclude, this study demonstrates the effectiveness of using engineering biology approaches to optimise allotopic expression of mitochondrial genes in the baker's yeast.

The translation of proteins with non-canonical amino acids (ncAAs) has emerged as a powerful technology for embedding new functional elements into proteins, enabling the development of novel enzymes, materials, and biopharmaceuticals. However, the utility of this approach has been hindered by weak translation efficiencies. To address this challenge, we sought to substantially improve orthogonal translation in Saccharomyces cerevisiae . We first evaluated recently described ΔNPylRS-class pyrrolysyl-tRNA synthetase systems and identified a homolog with ∼5.4-fold higher activity than the best previously reported pyrrolysyl system. Building on this advance, we leveraged the SCRaMbLE system in the semi-synthetic yeast strain Syn6.5 to generate structural genomic variation, and identified strains with enhanced ncAA incorporation. Pooling genomic alterations across enhanced strains, we identified an association for the deletion of several ribosomal protein genes with the increased production of ncAA-containing protein. These findings demonstrate a previously unrecognized role for ribosomal proteins in enabling alternate genetic codes. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/Y00812X/1, BB/W014483/1 Engineering and Physical Sciences Research Council, https://ror.org/0439y7842, EP/Y023722/1, EP/Y024753/1, EP/V05967X/1

Synthetic biology holds immense promise to tackle key problems in resource use, environmental remediation, and human health care. However, comprehensive safety measures are lacking to employ engineered microorganisms in open-environment applications. Genetically encoded biocontainment systems may solve this issue. Here, we describe such a system based on conditional stability of essential proteins. We used a destabilizing domain degron stabilized by estradiol addition (ERdd). We ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110 , DIS3 and RRP46 , were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of <5×10 −7 . Removal of its C-terminal domain decreased the escape frequency further to <10 −8 . Being based on conditional protein stability, the presented approach is mechanistically orthogonal to previously reported genetic biocontainment systems.

Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue Nδ-methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRSMIFAF) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus (MaPylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRSMIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRSMIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future. The Metschnikowia PUL1 and PUL2 genes induce pulcherrimin production in Saccharomyces cerevisiae, with antagonistic activity against Botrytis.

Technologies to profoundly engineer biology are becoming increasingly affordable, powerful, and accessible to a widening group of actors. While offering tremendous potential to fuel biological research and the bioeconomy, this development also increases the risk of inadvertent or deliberate creation and dissemination of pathogens. Effective regulatory and technological frameworks need to be developed and deployed to manage these emerging biosafety and biosecurity risks. Here, we review digital and biological approaches of a range of technology readiness levels suited to address these challenges. Digital sequence screening technologies already are used to control access to synthetic DNA of concern. We examine the current state of the art of sequence screening, challenges and future directions, and environmental surveillance for the presence of engineered organisms. As biosafety layer on the organism level, we discuss genetic biocontainment systems that can be used to created host organisms with an intrinsic barrier against unchecked environmental proliferation.

The synthetic yeast, Sc2.0, is nearing completion as consolidation of all 17 synthetic chromosomes into a single cell advances. This organism will be the first synthetic eukaryote and provides a highly plastic biological chassis built from the bottom-up using principles of biological design. This synthetic approach to genome construction has allowed the genetic code to be re-wired in this background to liberate the amber stop codon as a dedicated triplet for encoding non-canonical amino acids. The availability of an expanded set of amino acid building blocks allows precise control of protein structure and function, providing new opportunities to develop protein-based therapeutics, materials and catalysts. In this article, we review the challenges facing genetic code expansion research in yeast and highlight how the development of Sc2.0 provides new and exciting opportunities to address existing limitations.

The ability to redesign and reconstruct a cell at whole-genome level provides new platforms for biological study. The international synthetic yeast genome project-Sc2.0, designed by interrogating knowledge amassed by the yeast community to date, exemplifies how a classical synthetic biology "design-build-test-learn" engineering cycle can effectively test hypotheses about various genome fundamentals. The genome reshuffling SCRaMbLE system implemented in synthetic yeast strains also provides unprecedented diversified resources for genotype-phenotype study and yeast metabolic engineering. Further development of genome synthesis technology will shed new lights on complex biological processes in higher eukaryotes.

Computer-aided design (CAD) for synthetic biology promises to accelerate the rational and robust engineering of biological systems. It requires both detailed and quantitative mathematical and experimental models of the processes to (re)design biology, and software and tools for genetic engineering and DNA assembly. Ultimately, the increased precision in the design phase will have a dramatic impact on the production of designer cells and organisms with bespoke functions and increased modularity. CAD strategies require quantitative models of cells that can capture multiscale processes and link genotypes to phenotypes. Here, we present a perspective on how whole-cell, multiscale models could transform design-build-test-learn cycles in synthetic biology. We show how these models could significantly aid in the design and learn phases while reducing experimental testing by presenting case studies spanning from genome minimization to cell-free systems. We also discuss several challenges for the realization of our vision. The possibility to describe and build whole-cells in silico offers an opportunity to develop increasingly automatized, precise and accessible CAD tools and strategies.

Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.

For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness.

We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ(70) dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a 'forward' gene interferes with the expression of a 'reverse' gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.

The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-β), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for in vitro research using 3D high-density culture systems.

Light-switchable proteins offer numerous opportunities as tools for manipulating biological systems with exceptional degrees of spatiotemporal control. Most designed light-switchable proteins currently in use have not been optimised using the randomisation and selection/screening approaches that are widely used in other areas of protein engineering. Here we report an approach for screening light-switchable DNA-binding proteins that relies on light-dependent repression of the transcription of a fluorescent reporter. We demonstrate that the method can be used to recover a known light-switchable DNA-binding protein from a random library.

Background and Aims: While primary human hepatocytes are suitable for in vitro research on liver cells, their use for clinical application in liver support systems is problematic due to their limited availability.Within the European project d-LIVER a rat pancreatic cell line (B-13) that has been shown to be able to trans-differentiate into hepatocyte-like cells (B-13/H) when treated with dexamethasone is under investigation.These cells express hepatocyte specific markers and liver-specific CYP enzyme activities in their B-13/H phenotype.However, to apply such a cell source for extracorporeal liver support in patients, cells have to be expanded and processed in a closed environment and in a culture vessel that allows for culturing sufficient cell numbers to support a human liver.In these studies, an experimental 3D multi-compartment bioreactor system offering dynamic 3D culture conditions has been examined for its ability to support B-13/H function.Methods: Two different approaches were investigated in successive experiments.In the first approach, B-13 cells were inoculated and trans-differentiated in the bioreactor.In the second approach, trans-differentiated cells (B-13/H) were inoculated and maintained over 15 days in the bioreactor.Primary rat hepatocytes were used as a control culture and treated with the dexamethasone as for B-13 cells.Efficacy of trans-differentiation and cell viability were assessed by determination of liver-specific marker expression and function.With respect to the planned clinical use of the bioreactor system, a focus was laid on clinically relevant parameters.Results: Initial studies showed successful growth and transdifferentiation of B-13 cells in small-scale bioreactors.Typical hepatocyte functions, including CYP enzyme activities were observed.Urea production was comparable to primary hepatocytes.With respect to clinical application in extracorporeal liver support, further optimization of culture conditions and up-scaling of the methodology for larger cell numbers is under investigation. Conclusions:The results show that the 3D bioreactor system is suitable for culture and hepatic differentiation of the rat pancreatic progenitor cell line B-13.If a cell line of human origin with similar characteristics could be generated, it would be a promising cell source for bio-artificial liver support.

The rat pancreatic “B-13” acinar cell is a stable progenitor cell line that differentiates into hepatocyte-like cells (B-13/H cells) in 2D un-coated plastic culture with simple culture media in response to glucocorticoid exposure. Examination of cytochrome P450 indicated that the expression of a range of genes were similar to freshly isolated hepatocytes and that these gene products were functional on the basis of spectrophotometrically-detectable reduced carbon-monoxide haemoprotein and metabolism of several drugs. Since normal hepatocytes readily de-differentiate under similar conditions, we hypothesized that B-13 cells have undergone a variety of alterations that stabilise a progenitor phenotype and restrict differentiation to hepatocytes only (which if capitulated in human cells, could generate a readily accessible supply of functional human hepatocytes in vitro). To examine this hypothesis, the B-13 karyotype; pluripotency-inducing transcription factor expression and forced over-expression of these factors in B-13 cells were examined. B-13 cells were also injected into NOD/SCID mice and engraftment and differentiation assessed by RT-PCR, Western blotting, immunohistochemistry and fluorescent in situ hybridization (FISH). B-13 cells expressed four pluripotency-inducing transcription factors c-Myc, Klf4, Oct4 and Sox2 with only c-Myc expression maintained after glucocorticoid treatment. Over-expression of the pluripotency-inducing transcription factors blocked B-13/H formation in response to glucocorticoid. Injection of B-13 cells into NOD/SCID mice resulted in their engraftment to the pancreas and liver, with restricted differentiation to hepatocytes in the liver. The cells did not engraft to any other tissues examined. The ability of B-13 cells to specifically generate functional hepatocytes in vitro in response to glucocorticoid is therefore associated with genetic rearrangements that may facilitate expression of genes associated with plasticity (without leading to pluripotency), which are repressed by glucocorticoid treatment.

Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver.

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions.