Biology subjects

Al Nahas, K.

Publications and source records attributed to Al Nahas, K..

7 recordsLinked to original sources

bioRxiv2026

Automated Synthetic Cell-based Screening for Designed Proteins with Emergent Functions

Designing minimal biological systems with emergent functions such as spatiotemporal self-organization is a central goal of bottom-up synthetic biology. While computational optimization and design show promises to accelerate functional protein engineering through Design-Build-Test-Learn cycles, screening libraries for complex functions remains a major challenge. Conventional screens typically lack the spatiotemporal resolution and cell-like confinement required in bottom-up synthetic biology. Here, we present PUREdrop, an automated microfluidic platform that encapsulates and expresses protein libraries in thousands of picolitre-sized synthetic cells per construct. These droplets are sorted into a 96-well plate and analyzed by time-lapse imaging, allowing parallel quantification of expression kinetics and emergent functions. To demonstrate the platforms potential, we first screened computationally re-designed variants of the bacterial cell division protein FtsZ, identifying variants with improved bundling phenotypes and faster kinetics. We then extended our screening procedure towards general protein modulators of FtsZ and identified a combination that anchors filaments to the interface, producing a ring-like phenotype. PUREdrop bridges computational protein engineering and synthetic cell research, elevating the rational engineering of complex biological function to the next level.

synthetic biology↗

bioRxiv2021

A microfluidic platform for sequential assembly and separation of synthetic cell models

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and by-products, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a stream of vesicles suspension is partitioned into three fractions - purified GUVs, residual components, and a washing solution. Using our purification approach, we showed that giant vesicles can be separated from various residues - that range in size and chemical composition - with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

synthetic biology↗

bioRxiv2021

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, peptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions - their membranes. However, suitable assays to quantify the efficacy of peptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of peptide antibiotics. We reveal strong correlations between each peptides physicochemical activity at the membrane level and biological activity at the cellular level by assaying the membranolytic activities of the antibiotics on hundreds of individual giant lipid vesicles, and quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

microbiology↗

bioRxiv2021

Measuring thousands of single vesicle leakage events reveals the mode of action of antimicrobial peptides

Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, existing characterization assays lack the resolution necessary to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations, and related the observed modes with the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner, and for assigning specific modes of action to the corresponding antimicrobial mechanisms.

biophysics↗

bioRxiv2019

An integrated microfluidic platform for quantifying drug permeation across biomimetic vesicle membranes

The low membrane permeability of candidate drug molecules is a major challenge in drug development and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging since one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological salt and pH levels, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin across biomimetic liposomes.

biophysics↗

bioRxiv2018

Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner

Bacterial cells are critically dependent upon pH regulation. Most proteins function over a limited pH range and the pH gradient across the bacterial cell membrane is central to energy production and transduction1. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of E. coli. Indole is an aromatic molecule with diverse signalling roles that in bacteria is produced from tryptophan by the enzyme tryptophanase (TnaA)2. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry3,4. We show that E. coli cells growing under conditions where no indole is produced maintain their cytoplasmic pH at 7.8 {+/-} 0.2. In contrast, under conditions permitting indole production, pH is maintained at 7.2 {+/-} 0.2. Experiments where indole was added experimentally to non-producing cultures showed that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, the application of an artificial pulse of either of two non-biological proton ionophores (DNP or CCCP) caused a similar effect, suggesting that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane5. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a \"memory\" to maintain the correct cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.

microbiology↗

bioRxiv2017

Outperforming nature: synthetic enzyme built from DNA flips lipids of biological membranes at record rates

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membranes inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and single-molecule experiments we find the lipid transport rate catalyzed by the DNA nanostructure to exceed 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.

biophysics↗