Dynamic colocalization of 2 simultaneously active VSG expression sites within a single expression-site body in Trypanosoma brucei.

Monoallelic exclusion ensures that the African trypanosome Trypanosoma brucei exclusively expresses only 1 of thousands of different variant surface glycoprotein (VSG) coat genes. The active VSG is transcribed from 1 of 15 polycistronic bloodstream-form VSG expression sites (ESs), which are controlled in a mutually exclusive fashion. Unusually, T. brucei uses RNA polymerase I (Pol I) to transcribe the active ES, which is unprecedented among eukaryotes. This active ES is located within a unique extranucleolar Pol I body called the expression-site body (ESB). A stringent restriction mechanism prevents T. brucei from expressing multiple ESs at the same time, although how this is mediated is unclear. By using drug-selection pressure, we generated VSG double-expresser T. brucei lines, which have disrupted monoallelic exclusion, and simultaneously express 2 ESs in a dynamic fashion. The 2 unstably active ESs appear epigenetically similar to fully active ESs as determined by using chromatin immunoprecipitation for multiple epigenetic marks (histones H3 and H1, TDP1, and DNA base J). We find that the double-expresser cells, similar to wild-type single-expresser cells, predominantly contain 1 subnuclear ESB, as determined using Pol I or the ESB marker VEX1. Strikingly, simultaneous transcription of the 2 dynamically transcribed ESs is normally observed only when the 2 ESs are both located within this single ESB. This colocalization is reversible in the absence of drug selection. This discovery that simultaneously active ESs dynamically share a single ESB demonstrates the importance of this unique subnuclear body in restricting the monoallelic expression of VSG.

Identification of the ISWI Chromatin Remodeling Complex of the Early Branching Eukaryote Trypanosoma brucei.

ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution.

Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria.

Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs.

NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides.

Bioactive peptides are key molecules in health and medicine. Deep learning holds a big promise for the discovery and design of bioactive peptides. Yet, suitable experimental approaches are required to validate candidates in high throughput and at low cost. Here, we established a cell-free protein synthesis (CFPS) pipeline for the rapid and inexpensive production of antimicrobial peptides (AMPs) directly from DNA templates. To validate our platform, we used deep learning to design thousands of AMPs de novo. Using computational methods, we prioritized 500 candidates that we produced and screened with our CFPS pipeline. We identified 30 functional AMPs, which we characterized further through molecular dynamics simulations, antimicrobial activity and toxicity. Notably, six de novo-AMPs feature broad-spectrum activity against multidrug-resistant pathogens and do not develop bacterial resistance. Our work demonstrates the potential of CFPS for high throughput and low-cost production and testing of bioactive peptides within less than 24 h.

TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei.

The unicellular eukaryote Trypanosoma brucei is unusual in having very little transcriptional control. The bulk of the T. brucei genome is constitutively transcribed by RNA polymerase II (Pol II) as extensive polycistronic transcription units. Exceptions to this rule include several RNA Pol I transcription units such as the VSG expression sites (ESs), which are mono-allelically expressed. TbISWI, a member of the SWI2/SNF2 related chromatin remodeling ATPases, plays a role in repression of Pol I-transcribed ESs in both bloodstream- and procyclic-form T. brucei. We show that TbISWI binds both active and silent ESs but is depleted from the ES promoters themselves. TbISWI knockdown results in an increase in VSG transcripts from the silent VSG ESs. In addition to its role in the repression of the silent ESs, TbISWI also contributes to the downregulation of the Pol I-transcribed procyclin loci, as well as nontranscribed VSG basic copy arrays and minichromosomes. We also show that TbISWI is enriched at a number of strand switch regions which form the boundaries between Pol II transcription units. These strand switch regions are the presumed sites of Pol II transcription initiation and termination and are enriched in modified histones and histone variants. Our results indicate that TbISWI is a versatile chromatin remodeler that regulates transcription at multiple Pol I loci and is particularly abundant at many Pol II transcription boundaries in T. brucei.

Cell-Free Biosensors and AI Integration.

Cell-free biosensors hold a great potential as alternatives for traditional analytical chemistry methods providing low-cost low-resource measurement of specific chemicals. However, their large-scale use is limited by the complexity of their development.In this chapter, we present a standard methodology based on computer-aided design (CAD ) tools that enables fast development of new cell-free biosensors based on target molecule information transduction and reporting through metabolic and genetic layers, respectively. Such systems can then be repurposed to represent complex computational problems, allowing defined multiplex sensing of various inputs and integration of artificial intelligence in synthetic biological systems.

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates.

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts.

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

PeroxiHUB: A Modular Cell-Free Biosensing Platform Using H2O2 as Signal Integrator.

Cell-free systems have great potential for delivering robust, inexpensive, and field-deployable biosensors. Many cell-free biosensors rely on transcription factors responding to small molecules, but their discovery and implementation still remain challenging. Here we report the engineering of PeroxiHUB, an optimized H2O2-centered sensing platform supporting cell-free detection of different metabolites. H2O2 is a central metabolite and a byproduct of numerous enzymatic reactions. PeroxiHUB uses enzymatic transducers to convert metabolites of interest into H2O2, enabling rapid reprogramming of sensor specificity using alternative transducers. We first screen several transcription factors and optimize OxyR for the transcriptional response to H2O2 in a cell-free system, highlighting the need for preincubation steps to obtain suitable signal-to-noise ratios. We then demonstrate modular detection of metabolites of clinical interest─lactate, sarcosine, and choline─using different transducers mined via a custom retrosynthesis workflow publicly available on the SynBioCAD Galaxy portal. We find that expressing the transducer during the preincubation step is crucial for optimal sensor operation. We then show that different reporters can be connected to PeroxiHUB, providing high adaptability for various applications. Finally, we demonstrate that a peroxiHUB lactate biosensor can detect endogenous levels of this metabolite in clinical samples. Given the wide range of enzymatic reactions producing H2O2, the PeroxiHUB platform will support cell-free detection of a large number of metabolites in a modular and scalable fashion.

The automated Galaxy-SynBioCAD pipeline for synthetic biology design and engineering.

Here we introduce the Galaxy-SynBioCAD portal, a toolshed for synthetic biology, metabolic engineering, and industrial biotechnology. The tools and workflows currently shared on the portal enables one to build libraries of strains producing desired chemical targets covering an end-to-end metabolic pathway design and engineering process from the selection of strains and targets, the design of DNA parts to be assembled, to the generation of scripts driving liquid handlers for plasmid assembly and strain transformations. Standard formats like SBML and SBOL are used throughout to enforce the compatibility of the tools. In a study carried out at four different sites, we illustrate the link between pathway design and engineering with the building of a library of E. coli lycopene-producing strains. We also benchmark our workflows on literature and expert validated pathways. Overall, we find an 83% success rate in retrieving the validated pathways among the top 10 pathways generated by the workflows.

Toehold switch based biosensors for sensing the highly trafficked rosewood Dalbergia maritima.

Nucleic acid sensing is a 3 decades old but still challenging area of application for different biological sub-domains, from pathogen detection to single cell transcriptomics analysis. The many applications of nucleic acid detection and identification are mostly carried out by PCR techniques, sequencing, and their derivatives used at large scale. However, these methods' limitations on speed, cost, complexity and specificity have motivated the development of innovative detection methods among which nucleic acid biosensing technologies seem promising. Toehold switches are a particular class of RNA sensing devices relying on a conformational switch of secondary structure induced by the pairing of the detected trigger RNA with a de novo designed synthetic sensing mRNA molecule. Here we describe a streamlined methodology enabling the development of such a sensor for the RNA-mediated detection of an endangered plant species in a cell-free reaction system. We applied this methodology to help identify the rosewood Dalbergia maritima, a highly trafficked wood, whose protection is limited by the capacity of the authorities to distinguish protected logs from other unprotected but related species. The streamlined pipeline presented in this work is a versatile framework enabling cheap and rapid development of new sensors for custom RNA detection.

Optimising protein synthesis in cell-free systems, a review.

Over the last decades, cell-free systems have been extensively used for in vitro protein expression. A vast range of protocols and cellular sources varying from prokaryotes and eukaryotes are now available for cell-free technology. However, exploiting the maximum capacity of cell free systems is not achieved by using traditional protocols. Here, what are the strategies and choices one can apply to optimise cell-free protein synthesis have been reviewed. These strategies provide robust and informative improvements regarding transcription, translation and protein folding which can later be used for the establishment of individual best cell-free reactions per lysate batch.

Salicylic acid and thiourea mitigate the salinity and drought stress on physiological traits governing yield in pearl millet- wheat.

Plant growth is often affected with hampered physiological and cellular functioning due to salinity and drought stress. To assess the effectiveness of plant bioregulators (PBRs) in mitigating abiotic stresses, a double spilt plot field study was conducted with three replications at ICAR-CSSRI, research farm, Nain, Panipat. The study comprised of three deficit irrigation regimes viz., 100, 80 and 60% of crop evapo-transpiration (ETc) (I1, I2 and I3), four levels of irrigation water salinity i.e. 2, 4, 8, 12 dS m-1 (S0, S1, S2 and S3) and two PBRs salicylic acid (SA; G1) and thiourea (TU; G2). Irrigations, as per regimes and salinity, were applied at identified critical stages of wheat and if needed in pearl millet. PBRs were applied as seed priming and foliar sprays at two sensitive stages of respective crops. The trend of plant height, and physiological and biochemical traits was similar under different treatments at both stages, but differed significantly only at reproductive stage. Water deficit caused significant reduction in pearl millet (5.1%) and wheat (6.7%) grain yields. The reduction in grain yield under 8 and 12 dS m-1 was 12.90 and 22.43% in pearl millet and 7.68 and 32.93% in wheat, respectively compared to 2 dS m-1. Application of either SA (G1) or TU (G2) significantly enhanced plant height and grain yield, but magnitude of the increment was higher with SA in pearl millet and with TU in wheat. Application of SA and TU increased grain yield by 14.42 and 12.98 in pearl millet, and 12.90 and 17.36% in wheat, respectively. The plant height, RWC, TC, MI, LP, proline, Fv/Fm and Na/K ratio significantly reduced by salinity stress in pearl millet and both water and salinity stress in wheat. Application of both PBRs proved beneficial to mitigate adverse effect of water deficit and salt stress by significantly improving physiological traits, biochemical traits and ultimately grain yield in both crops.

Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics.

CRISPR guide RNAs (gRNAs) can be programmed with relative ease to allow the genetic editing of nearly any DNA or RNA sequence. Here, we propose novel molecular architectures to achieve RNA-dependent modulation of CRISPR activity in response to specific RNA molecules. We designed and tested, in both living Escherichia coli cells and cell-free assays for rapid prototyping, cis-repressed RNA-interacting guide RNA (igRNA) that switch to their active state only upon interaction with small RNA fragments or long RNA transcripts, including pathogen-derived mRNAs of medical relevance such as the human immunodeficiency virus infectivity factor. The proposed CRISPR-igRNAs are fully customizable and easily adaptable to the majority if not all the available CRISPR-Cas variants to modulate a variety of genetic functions in response to specific cellular conditions, providing orthogonal activation and increased specificity. We thereby foresee a large scope of application for therapeutic, diagnostic, and biotech applications in both prokaryotic and eukaryotic systems.

Metabolic perceptrons for neural computing in biological systems.

Synthetic biological circuits are promising tools for developing sophisticated systems for medical, industrial, and environmental applications. So far, circuit implementations commonly rely on gene expression regulation for information processing using digital logic. Here, we present a different approach for biological computation through metabolic circuits designed by computer-aided tools, implemented in both whole-cell and cell-free systems. We first combine metabolic transducers to build an analog adder, a device that sums up the concentrations of multiple input metabolites. Next, we build a weighted adder where the contributions of the different metabolites to the sum can be adjusted. Using a computational model fitted on experimental data, we finally implement two four-input perceptrons for desired binary classification of metabolite combinations by applying model-predicted weights to the metabolic perceptron. The perceptron-mediated neural computing introduced here lays the groundwork for more advanced metabolic circuits for rapid and scalable multiplex sensing.

Functionalization of an Antisense Small RNA.

In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5' untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology.

Using RNA as Molecular Code for Programming Cellular Function.

RNA is involved in a wide-range of important molecular processes in the cell, serving diverse functions: regulatory, enzymatic, and structural. Together with its ease and predictability of design, these properties can lead RNA to become a useful handle for biological engineers with which to control the cellular machinery. By modifying the many RNA links in cellular processes, it is possible to reprogram cells toward specific design goals. We propose that RNA can be viewed as a molecular programming language that, together with protein-based execution platforms, can be used to rewrite wide ranging aspects of cellular function. In this review, we catalogue developments in the use of RNA parts, methods, and associated computational models that have contributed to the programmability of biology. We discuss how RNA part repertoires have been combined to build complex genetic circuits, and review recent applications of RNA-based parts and circuitry. We explore the future potential of RNA engineering and posit that RNA programmability is an important resource for firmly establishing an era of rationally designed synthetic biology.