Biology subjects

Di Ventura, B.

Publications and source records attributed to Di Ventura, B..

13 recordsLinked to original sources

bioRxiv2026

Computational screen of promoter configurations that robustly sense transcription factor dynamics

Transcription factors (TFs) respond to external stimuli with time-varying changes in activity or localization (TF dynamics), driving differential transcriptional programs. Previous studies indicated that TF dynamics can be decoded at the promoter level in eukaryotes, yet a systematic understanding of robust solutions is lacking. By computationally screening over 10,000 mathematical models of multi-state promoters with various forms of TF-mediated regulation, we identify robust configurations that selectively respond to sustained ("pulse filtering") or pulsatile ("pulse boosting") TF dynamics. Promoters that activate via intermediate states and have negatively regulated deactivation robustly perform pulse filtering. In contrast, robust pulse boosting is achieved by promoters with a TF-mediated refractory state that permits short activation and recovers between pulses. Bifunctional TFs that exert activator- and repressor-like regulation extend the design space for pulse boosting. These results reveal general principles by which promoters interpret TF dynamics and suggest strategies to engineer synthetic systems to exploit them. HighlightsO_LIComputational screen of over 10,000 promoter models identifies features that enable promoters to selectively respond to sustained ("pulse filtering") or pulsatile ("pulse boosting") transcription factor (TF) dynamics. C_LIO_LIPromoters that activate via intermediate states and have negatively regulated deactivation robustly perform pulse filtering. C_LIO_LIPromoters with TF-regulated refractoriness robustly perform pulse boosting. C_LIO_LIPromoters regulated by bifunctional TFs extend the design space for pulse boosting. C_LI

systems biology↗

bioRxiv2025

Machine learning guided engineering of conditional split inteins for regulated protein splicing in mammalian cells

Inteins are proteins that excise themselves from precursor proteins and connect the flanking polypeptides with a peptide bond. Split inteins consist of two independently translated fragments that must associate to become splice-competent. They can be used for diverse post-translational protein modifications. Using the ML Int&in algorithm, we predicted unnatural split sites in two of the fastest and most efficient split inteins, gp41-1 and NrdJ-1, to generate functional variants with fragments of reduced mutual affinity. We harness this feature to create conditional versions of these inteins by controlling the physical proximity of the intein fragments with a light-inducible heterodimerization system. The resulting light-activatable gp41-1 and NrdJ-1 inteins enabled blue light-dependent control of Cre recombinase activity in mammalian cells, which we exploited to spatially control apoptosis via localized expression of truncated BID (tBID) and caspase-8. This work highlights the versatility of Int&in for designing conditional inteins for precise spatiotemporal protein regulation.

synthetic biology↗

bioRxiv2025

Two-Photon Lithography-Fabricated Deterministic Lateral Displacement Microfluidic System for Efficient Minicell Purification in Cancer Therapy

Chromosome-less minicells, derived from aberrant polar division events of bacterial cells, have emerged as promising nanocarriers for targeted cancer drug delivery due to their unique characteristics. A major challenge in their purification process lies in effectively isolating such spherical minicells (<1 {micro}m) from their rod-shaped parental cells (1-10 {micro}m). This study investigates the use of Deterministic Lateral Displacement (DLD) microfluidic systems for minicell purification, leveraging Two-Photon Lithography (TPL) for the rapid prototyping of high-resolution designs optimized for this purpose. Under laminar flow conditions, we investigated key DLD design parameters including symmetric and asymmetric post gaps, outlet widths, dual post arrays, fluidic-resistance-optimized design. To enhance separation efficiency, we developed a two-stage microfluidic separation system combining a spiral inertial chip and an optimized DLD chip in series. Utilizing high-resolution TPL for chip fabrication of an inertial chip with 12 spirals and an asymmetric DLD chip with a 2 {micro}m downstream post gap, we achieved a separation efficiency of 94%. This high efficiency achieved using microfluidics for the separation of cells differing in both shape and size, demonstrates the potential of advanced microfluidic systems in cell sorting. TOCThis study presents the use of Deterministic Lateral Displacement (DLD) microfluidic systems, fabricated via high-resolution 3D printing, Two-Photon Lithography, to isolate minicells for targeted cancer drug delivery. By optimizing post geometries, array designs, fluidic resistance, and integrating a spiral inertial stage, the system achieves 94% separation efficiency. These advancements ensure high throughput, operational stability, and continuous sorting of bacterial cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/660580v1_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@696f37org.highwire.dtl.DTLVardef@4c2aaorg.highwire.dtl.DTLVardef@883752org.highwire.dtl.DTLVardef@e7f513_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Chromosome segregation in Escherichia coli involves DNA binding by the MinC-MinD complex

The Escherichia coli Min system, composed of the proteins MinC, MinD, and MinE, is crucial for directing septum formation at mid-cell. MinD has also been shown to bind DNA in a non-sequence-specific manner and to tether plasmid DNA to liposomes. A role for the Min system in chromosome segregation has been proposed, whereby repeated, transient membrane-tethering events mediated by MinD could help prevent backward movements of the duplicated chromosomes once entropic forces weaken. However, the contribution of the other Min proteins to DNA binding has remained unclear. Here, we demonstrate that DNA binding is strongly enhanced when MinD and MinC form a complex, while MinE and FtsZ (at high concentrations) interfere with this binding. We reveal a critical role for the linker connecting MinCs N- and C-terminal domains in both DNA binding and inhibition of FtsZ polymerization. E. coli strains expressing MinC linker variants from the native locus, in combination with the alpha subunit of HU tagged with GFP, display chromosomes that appear more spread throughout the cell. This phenotype could reflect reduced compaction, chromosome segregation defects, or a combination of both. These findings suggest a previously unrecognized role for the MinC linker in DNA binding and provide new insights into a potential physiological role of MinCD-DNA bnding in chromosome organization and/or dynamics.

microbiology↗

bioRxiv2023

Int&in: a machine learning-based web server for split site identification in inteins

Inteins are proteins that excise themselves out of host proteins and ligate the flanking polypeptides in an auto-catalytic process called protein splicing. They are gaining momentum in synthetic biology for their ability to post-translationally modify proteins of interest. In nature, inteins are either contiguous or split, in which case the two intein fragments must first form a complex for the splicing to occur. So far, heuristic methods have been employed whenever a new split site in an intein had to be identified. To make the process of split site identification in inteins faster, easier and less costly, we developed Int&in, a web server that uses a gaussian Naive Bayes machine learning model to predict active and inactive split sites with high accuracy. The model was trained on a data set generated by us and validated using a large diverse data set from the literature, resulting in an accuracy of 0.76. Int&in will facilitate the engineering of novel split inteins for applications in biotechnology and synthetic biology.

synthetic biology↗

bioRxiv2023

Identification of functional Npu DnaE and gp41-1 inteins split in three fragments

Inteins are special proteins that auto-catalytically carry out a protein splicing reaction. Due to their ability to post-translationally modify target proteins in vitro and in vivo, they are used in different applications, ranging from protein purification to the construction of Boolean logic gates. So far inteins have been found to be either encoded by a single gene (contiguous inteins) or by two separate ones (split inteins). Previously, it has been shown that the contiguous Ssp and Rma DnaB inteins and the split Npu DnaE intein could be artificially split in three fragments and retain functionality. Here we report the identification of novel split sites within the N-terminal fragments of the Npu DnaE and gp41-1 split inteins that lead to synthetic functional three-piece versions of these inteins. These variants contribute to the toolkit of three-piece inteins that could be used in biotechnological applications based on highly-fragmented inteins.

molecular biology↗

bioRxiv2022

patcHwork: A user-friendly pH sensitivity analysis web server for protein sequences and structures

pH regulates protein function and interactions by altering the charge of individual residues causing the loss or gain of intra-molecular non-covalent bonds, which may additionally lead to structural rearrangements. While tools to analyze residue-specific charge distribution of protein sequences and structures at a given pH exist, currently no tool is available to investigate non-covalent bond changes at two different pH values. In an effort to make protein pH sensitivity analysis more accessible to researchers without computational structural biology background, we developed patcHwork, a web server that combines the identification of amino acids undergoing a charge shift with the determination of affected non-covalent bonds at two user-defined pH values. At the sequence-only level, patcHwork applies the Henderson-Hasselbalch equation to determine pH-sensitive residues. When the 3D protein structure is available, patcHwork can be employed to gain a deeper mechanistic understanding of the effect of pH on a protein of interest. This is achieved using the PDB2PQR and PROPKA tools and non-covalent bond determination algorithms. A user-friendly interface allows visualizing pH-sensitive residues as well as affected salt bridges, hydrogen bonds and aromatic (pi-pi and cation-pi) interactions. Importantly, patcHwork can be used to identify patches, a new concept we propose of pH-sensitive residues in close proximity on the protein structure, which may have a major impact on function. We demonstrate the attractiveness of patcHwork studying experimentally investigated pH-sensitive proteins. (Access:https://patchwork.biologie.uni-freiburg.de/) Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/478804v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@df873eorg.highwire.dtl.DTLVardef@12815a4org.highwire.dtl.DTLVardef@a4d718org.highwire.dtl.DTLVardef@256c10_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

Temporal control of the integrated stress response by a stochastic molecular switch

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response, by integrating quantitative experiments with mathematical modeling, and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 mRNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

cell biology↗

bioRxiv2021

Requirements for mammalian promoters to decode transcription factor dynamics

In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode them. Combining optogenetics, deep learning-based image analysis and mathematical modeling, we find that decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Furthermore, we propose a theoretical mechanism based on phase separation that would allow a promoter to be activated better by pulsatile than sustained TF signals. These results provide an understanding on how TF dynamics are decoded in mammalian cells, which is important to develop optimal strategies to counteract disease conditions, and suggest ways to achieve multiplexing in synthetic pathways.

systems biology↗

bioRxiv2021

Expanding the SiMPl plasmid toolbox for use with spectinomycin/streptomycin

We recently developed the SiMPl plasmid toolbox, which is constituted by pairs of plasmids, generically indicated as pSiMPlx_N and pSiMPlx_C, which can be stably maintained in Escherichia coli with a single antibiotic x. The method exploits the split intein gp41-1 to reconstitute the enzyme conferring resistance towards the antibiotic x, whereby each enzyme fragment is expressed from one of the plasmids in the pair. pSiMPl plasmids are currently available for use with ampicillin, kanamycin, chloramphenicol, hygromycin and puromycin. Here we introduce another pair for use with spectinomycin/streptomycin broadening the application spectrum of the SiMPl toolbox. To find functional splice sites in aminoglycoside adenylyltransferase we apply a streamlined strategy looking exclusively at the flexibility of native cysteine and serine residues, which we first validated splitting the enzymes conferring resistance towards ampicillin, kanamycin, chloramphenicol and hygromycin. This strategy could be used in the future to split other enzymes conferring resistance towards antibiotics.

synthetic biology↗

bioRxiv2020

The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment

Nearly all bacteria are encased in a peptidoglycan cell wall, an essential crosslinked matrix of polysaccharide strands and short peptide stems. In the Gram-negative model organism Escherichia coli, more than forty cell wall synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to cell wall metabolism remains unclear due to significant apparent redundancy, particularly among the cell wall autolysins. E. coli produces three major LytC-type-N-acetylmuramoyl-L-alanine amidases, which share a role in separating the newly formed daughter cells during cytokinesis. Here, we reveal two of the three amidases exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB--and to a lesser extent, AmiC--activity. Combining computational and genetic analysis, we demonstrate that low pH-dependent stimulation of AmiB requires three periplasmic amidase activators: EnvC, NlpD, and YgeR. Altogether, our findings support overlapping, but not redundant, roles for the E. coli amidases in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity. IMPORTANCEPenicillin and related {beta}-lactam antibiotics targeting the bacterial cell wall synthesis are among the most commonly prescribed antimicrobials worldwide. However, rising rates of antibiotic resistance and tolerance jeopardize their continued clinical use. Development of new cell wall active therapeutics, including those targeting cell wall autolysins, has been stymied in part due to high levels of apparent enzymatic redundancy. In this study, we report a subset of E. coli amidases involved in cell separation during cell division are not redundant and instead are preferentially active during growth in distinct pH environments. Specifically, we discover E. coli amidases AmiB and AmiC are activated by acidic pH. Three semi-redundant periplasmic regulators--NlpD, EnvC, and YgeR--collectively mediate low pH-dependent stimulation of amidase activity. This discovery contributes to our understanding of how the cell wall remains robust across diverse environmental conditions and reveals opportunities for the development of condition-specific antimicrobial agents.

microbiology↗

bioRxiv2020

An inducible AraC that responds to blue light instead of arabinose

In Escherichia coli, the operon responsible for the catabolism of L-arabinose is regulated by the dimeric DNA-binding protein AraC. In the absence of L-arabinose, AraC binds to the distal I1 and O2 half-sites, leading to repression of the downstream PBAD promoter. In the presence of the sugar, the dimer changes conformation and binds to the adjacent I1 and I2 half-sites, resulting in the activation of PBAD. Here we engineer blue light-inducible AraC dimers in Escherichia coli (BLADE) by swapping the dimerization domain of AraC with blue light-inducible dimerization domains. Using BLADE to overexpress proteins important for cell shape and division site selection, we reversibly control cell morphology with light. We demonstrate the exquisite light responsiveness of BLADE by employing it to create bacteriographs with an unprecedented quality. We then employ it to perform a medium-throughput characterization of 39 E. coli genes with poorly defined or completely unknown function. Finally, we expand the initial library and create a whole family of BLADE transcription factors (TFs), which we characterize using a novel 96-well light induction setup. Since the PBAD promoter is commonly used by microbiologists, we envisage that the BLADE TFs will bring the many advantages of optogenetic gene expression to the field of microbiology.

microbiology↗

bioRxiv2020

C-terminal eYFP fusion impairs Escherichia coli MinE function

The Escherichia coli Min system plays an important role in the proper placement of the septum ring (Z-ring) at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator together with the membrane-bound ATPase MinD, which results in MinD having a concentration gradient with maxima at the poles and minimum at mid-cell. MinC, the direct inhibitor of the Z-ring initiator protein FtsZ, forms a complex with MinD at the membrane, thus mirroring MinD polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. The existence of the oscillations was revealed by performing time-lapse microscopy with fluorescently-labeled Min proteins. These fusion proteins have been since then widely used to study properties of the Min system. Here we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo, in vitro and in silico approaches, we demonstrate that the eYFP compromises MinE ability to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. Taken together, our results indicate that this fusion is functionally impaired and should be used with caution in cell biological studies.

microbiology↗