SEARCH · bioRxiv Science

Results for “Synthetic Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,225 records · Page 68Linked to original sources

bioRxiv2024

Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.

microbiology↗

bioRxiv2024

Modeling reveals the strength of weak interactions in stacked ring assembly

Cells employ many large macromolecular machines for the execution and regulation of processes that are vital for cell and organismal viability. Interestingly, cells cannot synthesize these machines as functioning units. Instead, cells synthesize the molecular parts that must then assemble into the functional complex. Many important machines, including chaperones like GroEL and proteases like the proteasome, are comprised protein rings that are stacked on top of one another. While there is some experimental data regarding how stacked-ring complexes like the proteasome self-assemble, a comprehensive understanding of the dynamics of stacked ring assembly is currently lacking. Here, we developed a mathematical model of stacked trimer assembly, and performed an analysis of the assembly of the stacked homomeric trimer, which is the simplest stacked ring architecture. We found that stacked rings are particularly susceptible to a form of kinetic trapping that we term "deadlock," in which the system gets stuck in a state where there are many large intermediates that are not the fully-assembled structure, but that cannot productively react. When interaction affinities are uniformly strong, deadlock severely limits assembly yield. We thus predicted that stacked rings would avoid situations where all interfaces in the structure have high affinity. Analysis of available crystal structures indicated that indeed the majority - if not all - of stacked trimers do not contain uniformly strong interactions. Finally, to better understand the origins of deadlock, we developed a formal pathway analysis and showed that, when all the binding affinities are strong, many of the possible pathways are utilized. In contrast, optimal assembly strategies utilize only a small number of patwhays. Our work suggests that deadlock is a critical factor influencing the evolution of macromolecular machines, and provides general principles for not only understanding existing machines but also for the design of novel structures that can self-assemble efficiently. Statement of SignificanceUnderstanding the assembly macromolecular machines is important for understanding a wide range of cellular processes. Here, we use mathematical models to study the assembly of stacked rings, which are a common motif in these machines. Our models revealed that these complexes can readily get "stuck" during assembly when the binding affinity between subunits is too strong. This suggests an evolutionary pressure to favor weaker interactions, and our analysis of solved structures confirmed this prediction. Our findings not only contribute to the fundamental understanding of assembly but also offer insights into the evolutionary pressures shaping the architecture of stacked rings, and have implications for both cell and synthetic biology.

biophysics↗

bioRxiv2024

A meta-analysis of the gut microbiome in inflammatory bowel disease patients identifies disease-associated small molecules

Changes in the gut microbiome have been associated with several human diseases, but the molecular and functional details underlying these associations remain largely unknown. Here, we performed a multi-cohort analysis of small molecule biosynthetic gene clusters (BGCs) in 5,306 metagenomic samples of the gut microbiome from 2,033 Inflammatory Bowel Disease (IBD) patients and 833 matched healthy subjects and identified a group of Clostridia-derived BGCs that are significantly associated with IBD. Using synthetic biology, we discovered and solved the structures of six fatty acid amides as the products of the IBD-enriched BGCs. Using two mouse models of colitis, we show that the discovered small molecules disrupt gut permeability and exacerbate inflammation in chemically and genetically susceptible mice. These findings suggest that microbiome-derived small molecules may play a role in the etiology of IBD and represent a generalizable approach for discovering molecular mediators of microbiome-host interactions in the context of microbiome-associated diseases.

microbiology↗

bioRxiv2024

A modular toolkit for environmental Rhodococcus, Gordonia, and Nocardia enables complex metabolic manipulation.

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome, but largely lack genetic tools comparable to those available in model species such as E. coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208 and demonstrate conserved functionality in diverse environmental isolates of Rhodococcus, Nocardia and Gordonia. This toolkit encompasses Mycobacteriale origins of replication, broad-host range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux towards the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species. IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens. Rhodococcus, Gordonia and Nocardia are three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these, Rhodococcus ruber C208 was shown to degrade polyethylene, Gordonia paraffinivorans can assimilate carbon from solid hydrocarbons, and Nocardia neocaledoniensis (and many other Nocardia) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.

microbiology↗

bioRxiv2024

Thermostable in vitro transcription-translation for enzyme screening in microdroplets

BackgroundIn vitro expression involves the utilization of the transcription and translation machinery derived from the cell to produce one or more proteins of interest and has found widespread application in the optimization of gene circuits or metabolic pathways in synthetic biology but also in pharmaceutical manufacturing. Most in vitro expression systems available are active at moderate temperatures but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Moreover, given the fact that the main barrier toward the general use of in vitro expression is its high price compared with host-based recombinant expression, there is a need to develop alternative in vitro expression systems operating at high temperatures and compatible with technologies that enable ultrahigh-throughput screening in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. ResultsTo this end, we produced high-expression cell-free extracts from Thermus thermophilus for in vitro translation and supplemented them with thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable to other thermostable in vitro expression systems, while the preparation procedure is simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Although the composition of these extracts showed a high background in carboxyl esterase assays, {beta}-glucosidase and cellobiose hydrolase activities could be measured with minimal background. ConclusionsCell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

microbiology↗

bioRxiv2024

Chalkophomycin Biosynthesis Revealing Unique Enzyme Architecture for a Hybrid Nonribosomal Peptide Synthetase and Polyketide Synthase

Chalkophomycin is a novel chalkophore with antibiotic activities isolated from Streptomyces sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of N-hydroxylpyrrole, 2H-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable to coordinate cupric ion draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the chm biosynthetic gene cluster from S. sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The chm cluster was localized to a [~]31 kb DNA region, consisting of 19 open reading frames that encode five non-ribosomal peptide synthetase (ChmHIJLO), one modular polyketide synthases (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 chm-type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.

biochemistry↗

bioRxiv2024

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci

Gene replacement therapies primarily rely on adeno-associated viral (AAV) vectors for transgene expression. However, episomal expression can decline over time due to vector loss or epigenetic silencing. CRISPR-based integration methods offer promise for long-term transgene insertion. While the development of transgene integration methods has made substantial progress, identifying optimal insertion loci remains challenging. Skeletal muscle is a promising tissue for gene replacement owing to low invasiveness of intramuscular injections, relative proportion of body mass, the multinucleated nature of muscle, and the potential for reduced adverse effects. Leveraging endogenous promoters in skeletal muscle, we evaluated two high-expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts. We hijacked the muscle creatine kinase (Ckm) and myoglobin (Mb) promoters by co-delivering CRISPR-Cas9 and a donor plasmid with promoterless constructs encoding green fluorescent protein (GFP) or human Factor IX (hFIX). Additionally, we deeply profiled our genome and transcriptome outcomes from targeted integration and evaluated the safety of the proposed sites. This study introduces a proof-of-concept technology for achieving high-level therapeutic gene expression in skeletal muscle, with potential applications in targeted integration-based medicine and synthetic biology.

bioengineering↗

bioRxiv2024

Engineering of highly active and diverse nuclease enzymes by combining machine learning and ultra-high-throughput screening

Optimizing enzymes to function in novel chemical environments is a central goal of synthetic biology, but optimization is often hindered by a rugged, expansive protein search space and costly experiments. In this work, we present TeleProt, an ML framework that blends evolutionary and experimental data to design diverse protein variant libraries, and employ it to improve the catalytic activity of a nuclease enzyme that degrades biofilms that accumulate on chronic wounds. After multiple rounds of high-throughput experiments using both TeleProt and standard directed evolution (DE) approaches in parallel, we find that our approach found a significantly better top-performing enzyme variant than DE, had a better hit rate at finding diverse, high-activity variants, and was even able to design a high-performance initial library using no prior experimental data. We have released a dataset of 55K nuclease variants, one of the most extensive genotype-phenotype enzyme activity landscapes to date, to drive further progress in ML-guided design.

bioinformatics↗

bioRxiv2024

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating suicide exon and Cre recombinase

Chemical-inducible gene expression systems have been frequently used to regulate gene expression for functional genomics in various plant species. However, a convenient chemical-inducible system that can tightly regulate transgene expression in Nicotiana benthamiana is still missing. In this study, we developed a tightly regulated copper-inducible system that can be used to regulate transgene expression and perform cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system showed the least concerns of leakiness issues. Using the MoClo-based synthetic biology approach, we optimized the design of the copper-inducible system and incorporated the use of the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested NLRs and their matching AVRs, and it can also be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings provide new approaches for both fundamental and translational studies in plant functional genomics.

plant biology↗

bioRxiv2024

Long-read sequencing to detect full-length protein-protein interactions

Given the increased predictions on interactome size and demand for protein function information, methods for detecting protein-protein interactions remain a significant development area. The all-vs.-all sequencing (AVA-Seq) method utilizes a convergent fusion plasmid design to make two-hybrid technology amenable to next-generation sequencing. Here, we further innovate to take advantage of synthetic DNA technologies and Oxford Nanopore Technologies long-read sequencing improvements to allow us to determine full-length protein-protein interactions. Here, using this approach we recovered 159 protein-protein interactions from a set of 57 human proteins using multiple forms of validation. Further, when referencing a human gold standard set of interactions, eight full-length protein-protein interactions were recovered from an expected 28 interaction pairs (28.6%), a typical recovery rate for two-hybrid technologies. The AVA-Seq, in combination with the ease of synthetic DNA production and the MinION platform, offers a low-cost, high-throughput alternative for determining protein-protein interactions, which can be utilized in research labs at all stages. 3 Key PointsO_LIFirst application of long-read sequencing for full-length protein-protein interaction studies. C_LIO_LIThe recovery rate of the AVA-Seq method using full-length proteins is on par with other leading methods. C_LIO_LIAdvances in synthetic biology and sequencing technologies make full-length protein interactomes affordable and accessible. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/586447v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d81412org.highwire.dtl.DTLVardef@10dbb19org.highwire.dtl.DTLVardef@76a04borg.highwire.dtl.DTLVardef@15baf0_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Variable orthogonality of RDF-large serine integrase interactions within the phiC31 family

Large serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse, excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases ({phi}C31, {phi}BT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases - RDF pairs: {phi}C31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, {phi}C31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate {phi}C31 integrase. {phi}BT1 and TG1 RDFs were noticeably more effective than {phi}C31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase - RDF pair. The binding surface on RDF is much more conserved than the binding surface on integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase - RDF pairs. Additionally, the ability of a particular integrases preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches.

molecular biology↗

bioRxiv2024

Construction of whole cell bacterial biosensors as an alternative environmental monitoring technology to detect naphthenic acids in oil sands process-affected water.

After extraction of bitumen from oil sands deposits, the oil sand process-affected water (OSPW) is stored in tailings ponds. Naphthenic acids in tailings ponds have been identified as the primary contributor to toxicity to aquatic life. As an alternative to other analytical methods, here we identify bacterial genes induced after growth in naphthenic acids and use synthetic biology approaches to construct a panel of candidate biosensors for NA detection in water. The main promoters of interest were the atuAR promoters from a naphthenic acid degradation operon and upstream TetR regulator, the marR operon which includes a MarR regulator and downstream naphthenic acid resistance genes, and a hypothetical gene with a possible role in fatty acid biology. Promoters were printed and cloned as transcriptional lux reporter plasmids that were introduced into a tailings pond-derived Pseudomonas species. All candidate biosensor strains were tested for transcriptional responses to naphthenic acid mixtures and individual compounds. The three priority promoters respond in a dose-dependent manner, which allows semi-quantitative measurements, to simple, acyclic and complex NA mixtures, and each promoter has unique NA specificities. The limits of NA detection from the various NA mixtures ranged between 1.5 - 15 mg/L. The atuA and marR promoters also detected NA in small volumes of OSPW samples and were induced by extracts of the panel of OSPW samples. While biosensors have been constructed for other hydrocarbons, here we describe a biosensor approach that could be employed in environmental monitoring of naphthenic acids in oil sands mining wastewater.

microbiology↗

bioRxiv2024

3D-printing fabrication of microwave-microfluidic device for droplets network formation and characterisation

Microwave-microfluidic devices (MMDs) have emerged as precision tools for the rapid, accurate, sensitive, and non-invasive characterisation of low-volume liquids. However, the fabrication of MMDs remains a significant challenge due to the complexities associated with integrating fluidic ducts and electronic components. Herein, we present a versatile and economical 3D-printing approach for MMD fabrication, using liquid metal as an electrical conductor. Cyclic olefin copolymer, polylactic acid and polypropylene were identified as potential printable dielectric materials for MMD fabrication. 3D-printed cyclic olefin copolymer substrates exhibited the lowest loss tangent of 0.002 at 2.7GHz, making it an ideal material for high frequency engineering. Liquid metal, specifically gallium indium eutectic, was injected into the printed ducts to form conductive microwave structures. Exemplar MMDs were fabricated to integrate split-ring type microwave resonators and droplet-forming fluidic junctions. These devices were applied in the formation and characterisation of water-in-oil emulsions for constructing definable lipid-segregated droplet interface bilayer (DIB) networks. This work not only indicates the feasibility of using 3D-printing for rapid prototyping of customised MMDs but also demonstrates the potential of MMDs as a new research tool for biochemistry and synthetic biology.

bioengineering↗

bioRxiv2024

Optimised control of adaptive evolution with competing selective pressures

The development of methods to understand and control the population dynamics of microbial evolution remains an outstanding question in synthetic biology and biotechnology more broadly. Due to the stochastic nature of evolution, its limited observability, and complex intra-population dynamics, this presents a significant challenge. In this paper, we explore techniques to control the evolutionary dynamics of a population, based on manipulation of one or two orthogonal selective pressures, which may in turn be coupled to mutagenesis. Our approach builds on past research in evolutionary biology that developed frameworks to study intra-population variant competition during asexual adaptive processes (i.e. clonal interference). Extending this theory, we design optimal control strategies for one (or more) selective pressures that can be used to maximise the rate of adaptation across a population as a whole. We introduce a theoretical modelling framework for this process, which we support with both simulations and preliminary experimental data, providing a concrete basis for emerging control approaches to directed evolution and evolution-aware design.

evolutionary biology↗

bioRxiv2024

Development of a recombineering system for the acetogen Eubacterium limosum with Cas9 counterselection for markerless genome engineering

Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system allows recombination to reach an efficiency of up to 100%, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck Eubacterium limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/588731v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1366103org.highwire.dtl.DTLVardef@11b1f0borg.highwire.dtl.DTLVardef@1931600org.highwire.dtl.DTLVardef@1896e4c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

RiboDiffusion: Tertiary Structure-based RNA Inverse Folding with Generative Diffusion Models

RNA design shows growing applications in synthetic biology and therapeutics, driven by the crucial role of RNA in various biological processes. A fundamental challenge is to find functional RNA sequences that satisfy given structural constraints, known as the inverse folding problem. Computational approaches have emerged to address this problem based on secondary structures. However, designing RNA sequences directly from 3D structures is still challenging, due to the scarcity of data, the non-unique structure-sequence mapping, and the flexibility of RNA conformation. In this study, we propose RiboDiffusion, a generative diffusion model for RNA inverse folding that can learn the conditional distribution of RNA sequences given 3D backbone structures. Our model consists of a graph neural network-based structure module and a Transformer-based sequence module, which iteratively transforms random sequences into desired sequences. By tuning the sampling weight, our model allows for a trade-off between sequence recovery and diversity to explore more candidates. We split test sets based on RNA clustering with different cut-offs for sequence or structure similarity. Our model outperforms baselines in sequence recovery, with an average relative improvement of 11% for sequence similarity splits and 16% for structure similarity splits. Moreover, RiboDiffusion performs consistently well across various RNA length categories and RNA types. We also apply in-silico folding to validate whether the generated sequences can fold into the given 3D RNA backbones. Our method could be a powerful tool for RNA design that explores the vast sequence space and finds novel solutions to 3D structural constraints.

bioinformatics↗

bioRxiv2024

FAST-STEM: A human pluripotent stem cell engineering toolkit for rapid design-build-test-learn development of human cell-based therapeutic devices

Very recent clinical advances in stem cell derived tissue replacement and gene therapy, in addition to the rise of artificial intelligence-aided scientific discovery, have placed the possibility of sophisticated human cell-based therapies firmly within reach. However, development of such cells and testing of their engineered gene circuit components, has proven highly challenging, due to the need for generating stable cell lines for each design-build-test-learn engineering cycle. Current approaches to generating stable human induced pluripotent stem cell (hiPSC) lines are highly time-consuming and suffer from lack of control, poor integration efficiency, and limited functionality. Validation in clinically relevant stem cell derived tissues is also broadly lacking. Such drawbacks are prohibitive to repeatably conducting cutting-edge stem cell engineering with broad application within a realistic timeframe and will not scale with the future of regenerative medicine. We have developed FAST-STEM (Facile Accelerated Stem-cell Transgene integration with SynBio Tunable Engineering Modes), a hPSC engineering platform that drastically reduces the time to generate differentiation ready stem cell lines from several weeks to 5 days, exhibiting a ~612-fold improvement in transgene integration rate over previous methodologies. Additional FAST-STEM innovations include: (i) rapid and highly efficient transgene integration; (ii) copy number control; (iii) simultaneous or consecutive integration of multiple gene cassettes; (iv) library screen capability. In addition to this unique functional versatility, platform transportability and broad use case for stem cell-engineering was confirmed by differentiation into eight different cell types across nine different laboratories. This platform dramatically lowers the bar for integration of synthetic biology with regenerative medicine, enabling experiments which were previously deemed logistically impossible, thus paving the way for sophisticated human cell device development.

bioengineering↗

bioRxiv2024

Deciphering GB1's Single Mutational Landscape: Insights from MuMi Analysis

Mutational changes that affect the binding of the C2 fragment of Streptococcal protein G (GB1) to the Fc domain of human IgG (IgG-Fc) have been extensively studied using deep mutational scanning (DMS), and the binding affinity of all single mutations has been measured experimentally in the literature. To investigate the underlying molecular basis, we perform in-silico mutational scanning for all possible single mutations, along with 2-{micro}s-long molecular dynamics (WT-MD) of the wild-type (WT) GB1 in both unbound and IgG-Fc bound forms. We compute the hydrogen bonds between GB1 and IgG-Fc in WT-MD to identify the dominant hydrogen bonds for binding, which we then assess in conformations produced by Mutation and Minimization (MuMi) to explain the fitness landscape of GB1 and IgG-Fc binding. Furthermore, we analyze MuMi and WT-MD to investigate the dynamics of binding, focusing on the relative solvent accessibility (RSA) of residues and the probability of residues being located at the binding interface. With these analyses, we explain the interactions between GB1 and IgG-Fc and display the structural features of binding. Our findings pave the way for improved predictive accuracy in protein stability and interaction studies, which are crucial for advancements in drug design and synthetic biology.

biophysics↗