T7 phage-assisted evolution of riboswitches using error-prone replication and dual selection.

Leveraging riboswitches, non-coding mRNA fragments pivotal to gene regulation, poses a challenge in effectively selecting and enriching these functional genetic sensors, which can toggle between ON and OFF states in response to their cognate inducers. Here, we show our engineered phage T7, enabling the evolution of a theophylline riboswitch. We have replaced T7's DNA polymerase with a transcription factor controlled by a theophylline riboswitch and have created two types of host environments to propagate the engineered phage. Both types host an error-prone T7 DNA polymerase regulated by a T7 promoter along with another critical gene-either cmk or pifA, depending on the host type. The cmk gene is necessary for T7 replication and is used in the first host type for selection in the riboswitch's ON state. Conversely, the second host type incorporates the pifA gene, leading to abortive T7 infections and used for selection in the riboswitch's OFF state. This dual-selection system, termed T7AE, was then applied to a library of 65,536 engineered T7 phages, each carrying randomized riboswitch variants. Through successive passage in both host types with and without theophylline, we observed an enrichment of phages encoding functional riboswitches that conferred a fitness advantage to the phage in both hosts. The T7AE technique thereby opens new pathways for the evolution and advancement of gene switches, including non-coding RNA-based switches, setting the stage for significant strides in synthetic biology.

qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing.

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed Escherichia coli via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches.

Pseudotyping Bacteriophage P2 Tail Fibers to Extend the Host Range for Biomedical Applications.

Bacteriophages (phages) represent powerful potential treatments against antibiotic-resistant bacterial infections. Antibiotic-resistant bacteria represent a significant threat to global health, with an estimated 70% of infection-causing bacteria being resistant to one or more antibiotics. Developing novel antibiotics against the limited number of cellular targets is expensive and time-consuming, and bacteria can rapidly develop resistance. While bacterial resistance to phage can evolve, bacterial resistance to phage does not appear to spread through lateral gene transfer, and phage may similarly adapt through mutation to recover infectivity. Phages have been identified for all known bacteria, allowing the strain-selective killing of pathogenic bacteria. Here, we re-engineered the Escherichia coli phage P2 to alter its tropism toward pathogenic bacteria. Chimeric tail fibers formed between P2 and S16 genes were designed and generated through two approaches: homology- and literature-based. By presenting chimeric P2:S16 fibers on the P2 particle, our data suggests that the resultant phages were effectively detargeted from the native P2 cellular target, lipopolysaccharide, and were instead able to infect via the proteinaceous receptor, OmpC, the natural S16 receptor. Our work provides evidence that pseudotyping P2 is feasible and can be used to extend the host range of P2 to alternative receptors. Extension of this work could produce alternative chimeric tail fibers to target pathogenic bacterial threats. Our engineering of P2 allows adsorption through a heterologous outer-membrane protein without culturing in its native host, thus providing a potential means of engineering designer phages against pathogenic bacteria from knowledge of their surface proteome.

Scarless Recombineering of Phage in Lysogenic State.

We present a scarless recombineering-based method for introducing multiple point mutations into the genome of a temperate phage. The method uses the λ Red recombineering system to promote exogenous ssDNA oligos to anneal on the prophage lagging strand during host genome replication. DNA repair is suppressed by inducing the expression of a dominant-negative mutant protein of the methyl-directed mismatch repair system. Screening for recombinant cells without a selection marker is feasible due to its high recombination frequency, estimated as more than 40% after six cycles. The method enables scarless editing of the genome of a bacteriophage in 4-5 days.

Lambda Red Recombineering of Bacteriophage in the Lysogenic State.

We present a recombineering-based method for editing the genome of a temperate phage. The method uses the lambda Red recombination system to edit the genome of a lysogenized host with a prophage compatible with bacteriophage lambda. Linear DNA is used as the recombination substrate and antibiotic resistance is used as the basis for selection of recombinants. The method enables the genetic manipulation of a prophage in 3-5 days.

Engineering of a Promoter Repressed by a Light-Regulated Transcription Factor in Escherichia coli.

Light-regulated gene expression systems allow controlling gene expression in space and time with high accuracy. Contrary to previous synthetic light sensors that incorporate two-component systems which require localization at the plasma membrane, soluble one-component repression systems provide several advantageous characteristics. Firstly, they are soluble and able to diffuse across the cytoplasm. Secondly, they are smaller and of lower complexity, enabling less taxing expression and optimization of fewer parts. Thirdly, repression through steric hindrance is a widespread regulation mechanism that does not require specific interaction with host factors, potentially enabling implementation in different organisms. Herein, we present the design of the synthetic promoter PEL that in combination with the light-regulated dimer EL222 constitutes a one-component repression system. Inspired by previously engineered synthetic promoters and the Escherichia coli lacZYA promoter, we designed PEL with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound. PEL is repressed by EL222 under conditions of white light with a light-regulated repression ratio of five. Further, alternating conditions of darkness and light in cycles as short as one hour showed that repression is reversible. The design of the PEL-EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression systems. Finally, we compare the PEL-EL222 system with similar systems and suggest general improvements that could optimize and extend the functionality of EL222-based as well as other one-component repression systems.

Engineering Bacteria to Produce Pure Phage-like Particles for Gene Delivery.

Natural and engineered phages have been used in many applications, but their use to deliver user-defined genetic cargoes has been hampered by contamination with replicative phage, restricting use of the technology beyond the laboratory. Here we present a method to produce transducing particles without contamination. In addition, we demonstrate the use of a helper phage-free transducing particle preparation as an antimicrobial agent. This will pave the way for the development of new phage-based technologies with greater scope than lytic phage therapy.

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.

Reinforcement learning in synthetic gene circuits.

Synthetic gene circuits allow programming in DNA the expression of a phenotype at a given environmental condition. The recent integration of memory systems with gene circuits opens the door to their adaptation to new conditions and their re-programming. This lays the foundation to emulate neuromorphic behaviour and solve complex problems similarly to artificial neural networks. Cellular products such as DNA or proteins can be used to store memory in both digital and analog formats, allowing cells to be turned into living computing devices able to record information regarding their previous states. In particular, synthetic gene circuits with memory can be engineered into living systems to allow their adaptation through reinforcement learning. The development of gene circuits able to adapt through reinforcement learning moves Sciences towards the ambitious goal: the bottom-up creation of a fully fledged living artificial intelligence.

Accelerated evolution of a minimal 63-amino acid dual transcription factor.

Transcription factors control gene expression in all life. This raises the question of what is the smallest protein that can support such activity. In nature, Cro from bacteriophage λ is one of the smallest known repressors (66 amino acids), and activators are typically much larger (e.g., λ cI, 237 amino acids). Previous efforts to engineer a minimal activator from λ Cro resulted in no activity in vivo in cells. In this study, we show that directed evolution results in a new Cro activator-repressor that functions as efficiently as λ cI in vivo. To achieve this, we develop phagemid-assisted continuous evolution (PACEmid). We find that a peptide as small as 63 amino acids functions efficiently as an activator and/or repressor. To our knowledge, this is the smallest protein activator that enables polymerase recruitment, highlighting the capacity of transcription factors to evolve from very short peptide sequences.

Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7.

With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.

Engineering a Circular Riboregulator in Escherichia coli.

RNAs of different shapes and sizes, natural or synthetic, can regulate gene expression in prokaryotes and eukaryotes. Circular RNAs have recently appeared to be more widespread than previously thought, but their role in prokaryotes remains elusive. Here, by inserting a riboregulatory sequence within a group I permuted intron-exon ribozyme, we created a small noncoding RNA that self-splices to produce a circular riboregulator in Escherichia coli. We showed that the resulting riboregulator can trans-activate gene expression by interacting with a cis-repressed messenger RNA. We characterized the system with a fluorescent reporter and with an antibiotic resistance marker, and we modeled this novel posttranscriptional mechanism. This first reported example of a circular RNA regulating gene expression in E. coli adds to an increasing repertoire of RNA synthetic biology parts, and it highlights that topological molecules can play a role in the case of prokaryotic regulation.

Engineering CRISPR guide RNA riboswitches for in vivo applications.

CRISPR-based genome editing provides a simple and scalable toolbox for a variety of therapeutic and biotechnology applications. Whilst the fundamental properties of CRISPR proved easily transferable from the native prokaryotic hosts to eukaryotic and multicellular organisms, the tight control of the CRISPR-editing activity remains a major challenge. Here we summarise recent developments of CRISPR and riboswitch technologies and recommend novel functionalised synthetic-gRNA (sgRNA) designs to achieve inducible and spatiotemporal regulation of CRISPR-based genetic editors in response to cellular or extracellular stimuli. We believe that future advances of these tools will have major implications for both basic and applied research, spanning from fundamental genetic studies and synthetic biology to genetic editing and gene therapy.

Boolean Computation in Plants Using Post-translational Genetic Control and a Visual Output Signal.

Due to autotrophic growing capacity and extremely rich secondary metabolism, plants should be preferred targets of synthetic biology. However, developments in plants usually run below those in other taxonomic groups. In this work we engineered genetic circuits capable of logic YES, OR and AND Boolean computation in plant tissues with a visual output signal. The circuits, which are deployed by means of Agrobacterium tumefaciens, perform with the conditional activity of the MYB transcription factor Rosea1 from Antirrhinum majus inducing the accumulation of anthocyanins, plant endogenous pigments that are directly visible to the naked eye or accurately quantifiable by spectrophotometric analysis. The translational fusion of Rosea1 to several viral proteins, such as potyvirus NIb or fragments thereof, rendered the transcription factor inactive. However, anthocyanin accumulation could be restored by inserting protease cleavage sites between both moieties of the fusion and by coexpressing specific proteases, such as potyvirus nuclear inclusion a protease.

Modulation of Intracellular O2 Concentration in Escherichia coli Strains Using Oxygen Consuming Devices.

The use of cell factories for the production of bulk and value-added compounds is nowadays an advantageous alternative to the traditional petrochemical methods. Nevertheless, the efficiency and productivity of several of these processes can improve with the implementation of micro-oxic or anoxic conditions. In the industrial setting, laccases are appealing catalysts that can oxidize a wide range of substrates and reduce O2 to H2O. In this work, several laccase-based devices were designed and constructed to modulate the intracellular oxygen concentration in bacterial chassis. These oxygen consuming devices (OCDs) included Escherichia coli's native laccase (CueO) and three variants of this protein obtained by directed evolution. The OCDs were initially characterized in vitro using E. coli DH5α protein extracts and subsequently using extracts obtained from other E. coli strains and in vivo. Upon induction of the OCDs, no major effect on growth was observed in four of the strains tested, and analysis of the cell extract protein profiles revealed increased levels of laccase. Moreover, oxygen consumption associated with the OCDs occurred under all of the conditions tested, but the performance of the devices was shown to be strain-dependent, highlighting the importance of the genetic background even in closely related strains. One of the laccase variants showed 13- and 5-fold increases in oxidase activity and O2 consumption rate, respectively. Furthermore, it was also possible to demonstrate O2 consumption in vivo using l-DOPA as the substrate, which represents a proof of concept that these OCDs generate an intracellular oxygen sink, thereby manipulating the redox status of the cells. In addition, the modularity and orthogonality principles used for the development of these devices allow easy reassembly and fine-tuning, foreseeing their introduction into other chassis/systems.

Quantitative Characterization of Translational Riboregulators Using an in Vitro Transcription-Translation System.

Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.

Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters.

Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the 'green' Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P rnpB , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.

Engineering of biomolecules by bacteriophage directed evolution.

Conventional in vivo directed evolution methods have primarily linked the biomolecule's activity to bacterial cell growth. Recent developments instead rely on the conditional growth of bacteriophages (phages), viruses that infect and replicate within bacteria. Here we review recent phage-based selection systems for in vivo directed evolution. These approaches have been applied to evolve a wide range of proteins including transcription factors, polymerases, proteases, DNA-binding proteins, and protein-protein interactions. Advances in this field expand the possible applications of protein and RNA engineering. This will ultimately result in new biomolecules with tailor-made properties, as well as giving us a better understanding of basic evolutionary processes.