Contracting the Host Range of Bacteriophage T7 Using a Continuous Evolution System.

Bacteriophage T7 is an intracellular virus that recognizes its host via tail and tail fiber proteins known as receptor-binding proteins (RBPs). The RBPs attach to a specific lipopolysaccharide (LPS) displayed on the host. While there are various reports of phage host range expansion resulting from mutations in the RBP encoding genes, there is little evidence for contraction of host range. Notably, most experimental systems have not monitored changes in host range in the presence of several hosts simultaneously. Here, we use a continuous evolution system to show that T7 phages grown in the presence of five restrictive strains and one permissive host, each with a different LPS, gradually cease to recognize the restrictive strains. Remarkably, this result was obtained in experiments with six different permissive hosts. The altered specificity is due to mutations in the RBPs as determined by gene sequencing. The results of using this system demonstrate a major role for RBPs in restricting the range of futile infections, and this process can be harnessed to reduce the host range in applications such as recognition and elimination of a specific bacterial serotype by bacteriophages.

Combining a Base Deaminase Mutator with Phage-Assisted Evolution.

Phage-assisted evolution has emerged as a powerful technique for improving a protein's function by using mutagenesis and selective pressure. However, mutations typically occur throughout the host's genome and are not limited to the gene-of-interest (GOI): these undesirable genomic mutations can yield host cells that circumvent the system's selective pressure. Our system targets mutations specifically toward the GOI by combining T7 targeted mutagenesis and phage-assisted evolution. This system improves the structure and function of proteins by accumulating favorable mutations that can change its binding affinity, specificity, and activity.

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.

Joint Efforts of Replicative Helicase and SSB Ensure Inherent Replicative Tolerance of G-Quadruplex.

G-quadruplex (G4) is a four-stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single-molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging-strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single-stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading-strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

Making RNA: Using T7 RNA polymerase to produce high yields of RNA from DNA templates.

RNA is playing an ever-growing role in molecular biology and biomedicine due to the many ways it influences gene expression and its increasing use in modern therapeutics. Hence, production of RNA molecules in large quantity and high purity has become essential for advancing basic scientific research and for developing next-generation therapeutics. T7 RNA polymerase (RNAP) is a DNA-dependent RNA polymerase of bacteriophage origin and it is the most widely-utilized tool enzyme for producing RNA. Here we describe a set of robust methods for in vitro transcribing RNA molecules from DNA templates using T7 RNAP, along with a set of subsequent RNA purification schemes. In the first part of this chapter, we provide the general method for T7 RNAP-based in vitro transcription and technical notes for troubleshooting failed or inefficient transcription. We also provide modified protocols for preparing specialized RNA transcripts. In the second part, we provide two purification methods using either gel-based denaturing purification or size exclusion column-based non-denaturing purification for isolating high-purity RNA products from transcription reaction mixtures and preparing them for downstream applications. This chapter is designed to provide researchers with versatile ways to efficiently generate RNA molecules of interest and a troubleshooting guide should they encounter problems while working with in vitro transcription using T7 RNAP.

T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells.

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-ß receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Construction of a T7 phage random peptide library by combining seamless cloning with in vitro translation.

T7 phage libraries displaying random peptides are powerful tools for screening peptide sequences that bind to various target molecules. The T7 phage system has the advantage of less biased peptide distribution compared to the M13 phage system. However, the construction of T7 phage DNA is challenging due to its long 36 kb linear DNA. Furthermore, the diversity of the libraries depends strongly on the efficiency of commercially available packaging extracts. To address these issues, we examined the combination of seamless cloning with cell-free translation systems. Seamless cloning technologies have been widely used to construct short circular plasmid DNA, and several recent studies showed that cell-free translation can achieve more diverse phage packaging. In this study, we combined these techniques to construct four libraries (CX7C, CX9C, CX11C and CX13C) with different random regions lengths. The libraries thus obtained all showed diversity > 109 plaque forming units (pfu). Evaluating our libraries with an anti-FLAG monoclonal antibody yielded the correct epitope sequence. The results indicate that our libraries are useful for screening peptide epitopes against antibodies. These findings suggest that our system can efficiently construct T7 phage libraries with greater diversity than previous systems.

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain.

BACKGROUND: The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). RESULTS: Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. CONCLUSIONS: We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.

Development of an Efficient Recombinant Protein Expression System in Clostridium saccharoperbutylacetonicum Based on the Bacteriophage T7 System.

Recombinant proteins have broad applications. However, there is a lack of a recombinant protein expression system specifically for large-scale production in anaerobic hosts. Here, we developed a powerful and stringently inducible protein expression system based on the bacteriophage T7 system in the strictly anaerobic solvent-producing Clostridium saccharoperbutylacetonicum. With the integration of a codon optimized T7 RNA polymerase into the chromosome, a single plasmid carrying a T7 promoter could efficiently drive high-level expression of the target gene in an orthogonal manner, which was tightly regulated by a lactose-inducible system. Furthermore, by deleting beta-galactosidase genes involved in lactose metabolism, the transcriptional strength was further improved. In the ultimately optimized strain TM-07, the transcriptional strength of the T7 promoter showed 9.5-fold increase compared to the endogenous strong promoter Pthl. The heterologous NADP+-dependent 3-hydroxybutyryl-CoA dehydrogenase (Hbd1) from C. kluyveri was expressed in TM-07, and the yield of the recombinant protein reached 30.4-42.4% of the total cellular protein, surpassing the strong protein expression systems in other Gram-positive bacteria. The relative activity of Hbd1 in the crude enzyme was 198.0 U/mg, which was 8.3-fold higher than the natural activity in C. kluyveri. The relative activity of the purified enzyme reached 467.4 U/mg. To the best of our knowledge, this study represents the first application of the T7 expression system in Clostridium species, and this optimized expression system holds great potential for large-scale endotoxin-free recombinant protein production under strictly anaerobic conditions. This development paves the way for significant advancements in biotechnology and opens up new avenues for industrial applications.

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield.

Phage therapy to treat life-threatening drug-resistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of translation initiation factor IF-3 (infC) and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.

Regulation of T7 gp2.5 binding dynamics by its C-terminal tail, template conformation and sequence.

Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) binds to and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. We directly visualize fluorescently labelled T7 gp2.5 binding to ssDNA at the single-molecule level. Upon binding, T7 gp2.5 reduces the contour length of ssDNA by stacking nucleotides in a force-dependent manner, suggesting T7 gp2.5 suppresses the formation of secondary structure. Next, we investigate the binding dynamics of T7 gp2.5 and a deletion mutant lacking 21 C-terminal residues (gp2.5-Δ21C) under various template tensions. Our results show that the base sequence of the DNA molecule, ssDNA conformation induced by template tension, and the acidic terminal domain from T7 gp2.5 significantly impact on the DNA binding parameters of T7 gp2.5. Moreover, we uncover a unique template-catalyzed recycling behaviour of T7 gp2.5, resulting in an apparent cooperative binding to ssDNA, facilitating efficient spatial redistribution of T7 gp2.5 during the synthesis of successive Okazaki fragments. Overall, our findings reveal an efficient binding mechanism that prevents the formation of secondary structures by enabling T7 gp2.5 to rapidly rebind to nearby exposed ssDNA regions, during lagging strand DNA synthesis.

ASFV proteins presented at the surface of T7 phages induce strong antibody responses in mice.

African swine fever virus (ASFV) infection causes substantial economic losses to the swine industry worldwide, and there are still no safe and effective vaccines or therapeutics available. The granulated virus antigen improves the antigen present process and elicits high antibody reaction than the subunit antigen. In this study, the SpyTag peptide-p10 fusion protein was altered and displayed on the surface of the T7 phage to construct an engineered phage (T7-ST). At the same time, ASFV antigen-Spycatcher C-terminal-fused protein (antigen-SC) was expressed and purified by an E. coli prokaryotic expression system. Five virus-like particles (VLPs) displaying the main ASFV antigenic proteins P30, P54, P72, CD2v, and K145R were reconstructed by the isopeptide bond between SpyTag and antigen-SC proteins. The stability of five ASFV VLPs in high temperature and extreme pH conditions was evaluated by transmission electron microscopy (TEM) and plaque analysis. All ASFV VLPs induced a high titer antigen-specific antibody response in mice. Our results showed that the granulated antigen displaying ASFV protein on the surface of the T7 phage provides a robust potential vaccine and diagnostic tool to address the challenge of the ASFV pandemic.

Clamp Interactions with +3/+6 Duplex and Upstream-to-Downstream Allosteric Effects in Late Steps of Forming a Stable RNA Polymerase-Promoter Open Complex.

Stable 37 °C open complexes (OC) of E. coli RNA polymerase (RNAP) at λPR and T7A1 promoters form at similar rates but have very different lifetimes. To understand the downstream interactions responsible for OC lifetime, how promoter sequence directs them and when they form, we report lifetimes of stable OC and unstable late (I2) intermediates for promoters with different combinations of λPR (L) and T7A1 (T) discriminators, core promoters and UP elements. I2 lifetimes are similarly short, while stable OC lifetimes differ greatly, determined largely by the discriminator and modulated by core-promoter and UP elements. The free energy change ΔG3o for I2 â†’ stable OC is approximately -4 kcal more favorable for L-discriminator than for T-discriminator promoters. Downstream-truncation at +6 (DT+6) greatly destabilizes OC at L-discriminator but not T-discriminator promoters, making all ΔG3o values similar (approximately -4 kcal). Urea reduces OC lifetime greatly by affecting ΔG3o. We deduce that urea acts by disfavoring coupled folding of key elements of the ß'-clamp, that I2 is an open-clamp OC, and that clamp-closing in I2 â†’ stable OC involves coupled folding. Differences in ΔG3o between downstream-truncated and full-length promoters yield contributions to ΔG3o from interactions with downstream mobile elements (DME) including ß-lobe and ß'-jaw, more favorable for L-discriminator than for T-discriminator promoters. We deduce how competition between far-downstream DNA and σ70 region 1.1 affects ΔG3o values. We discuss variant-specific ΔG3o contributions in terms of the allosteric network by which differences in discriminator and -10 sequence are sensed and transmitted downstream to affect DME-duplex interactions in I2 â†’ stable OC.

Transcriptomics-Driven Characterization of LUZ100, a T7-like Pseudomonas Phage with Temperate Features.

Autographiviridae is a diverse yet distinct family of bacterial viruses marked by a strictly lytic lifestyle and a generally conserved genome organization. Here, we characterized Pseudomonas aeruginosa phage LUZ100, a distant relative of type phage T7. LUZ100 is a podovirus with a limited host range which likely uses lipopolysaccharide (LPS) as a phage receptor. Interestingly, infection dynamics of LUZ100 indicated moderate adsorption rates and low virulence, hinting at temperate characteristics. This hypothesis was supported by genomic analysis, which showed that LUZ100 shares the conventional T7-like genome organization yet carries key genes associated with a temperate lifestyle. To unravel the peculiar characteristics of LUZ100, ONT-cappable-seq transcriptomics analysis was performed. These data provided a bird's-eye view of the LUZ100 transcriptome and enabled the discovery of key regulatory elements, antisense RNA, and transcriptional unit structures. The transcriptional map of LUZ100 also allowed us to identify new RNA polymerase (RNAP)-promoter pairs that can form the basis for biotechnological parts and tools for new synthetic transcription regulation circuitry. The ONT-cappable-seq data revealed that the LUZ100 integrase and a MarR-like regulator (proposed to be involved in the lytic/lysogeny decision) are actively cotranscribed in an operon. In addition, the presence of a phage-specific promoter transcribing the phage-encoded RNA polymerase raises questions on the regulation of this polymerase and suggests that it is interwoven with the MarR-based regulation. This transcriptomics-driven characterization of LUZ100 supports recent evidence that T7-like phages should not automatically be assumed to have a strictly lytic life cycle. IMPORTANCE Bacteriophage T7, considered the "model phage" of the Autographiviridae family, is marked by a strictly lytic life cycle and conserved genome organization. Recently, novel phages within this clade have emerged which display characteristics associated with a temperate life cycle. Screening for temperate behavior is of utmost importance in fields like phage therapy, where strictly lytic phages are generally required for therapeutic applications. In this study, we applied an omics-driven approach to characterize the T7-like Pseudomonas aeruginosa phage LUZ100. These results led to the identification of actively transcribed lysogeny-associated genes in the phage genome, pointing out that temperate T7-like phages are emerging more frequent than initially thought. In short, the combination of genomics and transcriptomics allowed us to obtain a better understanding of the biology of nonmodel Autographiviridae phages, which can be used to optimize the implementation of phages and their regulatory elements in phage therapy and biotechnological applications, respectively.

Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage.

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase.

Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds-Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

Recombinant Protein Expression Chassis Library of Vibrio natriegens by Fine-Tuning the Expression of T7 RNA Polymerase.

Vibrio natriegens is the fastest-growing bacteria, and its doubling time is less than 10 min. At present, the T7 expression system has been introduced into V. natriegens for heterologous protein expression, including the commercial strain Vmax1 and the variant VnDX,2 which is a backup expression chassis of Escherichia coli BL21(DE3). However, the strength of the existing T7 expression system is not optimal for every recombinant protein. The different expression strengths of T7 RNA polymerase (T7 RNAP) can be obtained by changing the promoter and ribosome binding site (RBS) sequences of T7 RNAP at different transcription and translation levels. In this work, we obtained a robust VnDX variant library with the fine-tuning T7 RNAP using the industrially used enzyme glucose dehydrogenase (GDH) as the reporter protein. Among this library, the variant VnDX-tet, whose promoter of T7 RNAP was changed from PlacUV5 to Ptet, showed that the reporter enzyme GDH activity was increased by 109% by the T7 expression system. Similarly, variants with different T7 RNAP translation levels were obtained by changing RBS sequences upstream of T7 RNAP, and the results showed that the variant VnDX-RBS12/pGDH had the highest GDH activity, which increased by 12.6%. The VnDX variant library constructed in this study with different T7 expression strengths provides a choice for expressing various recombinant proteins, greatly expanding the application of V. natriegens.

Engineering of a T7 Bacteriophage Endolysin Variant with Enhanced Amidase Activity.

The therapeutic use of bacteriophage-encoded endolysins as enzybiotics has increased significantly in recent years due to the emergence of antibiotic resistant bacteria. Phage endolysins lyse the bacteria by targeting their cell wall. Various engineering strategies are commonly used to modulate or enhance the utility of therapeutic enzymes. This study employed a structure-guided mutagenesis approach to engineer a T7 bacteriophage endolysin (T7L) with enhanced amidase activity and lysis potency via replacement of a noncatalytic gating residue (His 37). Two H37 variants (H37A and H37K) were designed and characterized comprehensively using integrated biophysical and biochemical techniques to provide mechanistic insights into their structure-stability-dynamics-activity paradigms. Among the studied proteins, cell lysis data suggested that the obtained H37A variant exhibits amidase activity (∼35%) enhanced compared to that of wild-type T7 endolysin (T7L-WT). In contrast to this, the H37K variant is highly unstable, prone to aggregation, and less active. Comparison of the structure and dynamics of the H37A variant to those of T7L-WT evidenced that the alteration at the site of H37 resulted in long-range structural perturbations, attenuated the conformational heterogeneity, and quenched the microsecond to millisecond time scale motions. Stability analysis confirmed the altered stability of H37A compared to that of its WT counterpart. All of the obtained results established that the H37A variant enhances the lysis activity by regulating the stability-activity trade-off. This study provided deeper atomic level insights into the structure-function relationships of endolysin proteins, thus aiding researchers in the rational design of engineered endolysins with enhanced therapeutic properties.

An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts.

In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant.

[Editing of Phage Genomes - Recombineering-Assisted SpCas9 Modification of Model Coliphages T7, T5, and T3].

Bacteriophages-viruses that infect bacterial cells - are the most abundant biological entities on Earth. The use of phages in fundamental research and industry requires tools for precise manipulation of their genomes. Yet, compared to bacterial genome engineering, modification of phage genomes is challenging because of the lack of selective markers and thus requires laborious screenings of recombinant/mutated phage variants. The development of the CRISPR-Cas technologies allowed to solve this issue by the implementation of negative selection that eliminates the parental phage genomes. In this manuscript, we summarize current methods of phage genome engineering and their coupling with CRISPR-Cas technologies. We also provide examples of our successful application of these methods for introduction of specific insertions, deletions, and point mutations in the genomes of model Escherichia coli lytic phages T7, T5, and T3.