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Pasotti, L.

Publications and source records attributed to Pasotti, L..

5 recordsLinked to original sources

Engineered Living Materials for Bioproduction Downstream Processing

De novo engineered living materials (ELMs) are cellular systems that self-assemble into macroscopic structures through genetically encoded interactions, offering a route to programmable materials grown directly from living cells. Despite their promise, the molecular design principles that enable scalable self-assembly in de novo ELMs remain poorly understood. Here, we engineer Escherichia coli to display elastin-like polypeptides (ELPs) on the outer membrane, transforming single cells into self-assembling living materials through weak intermolecular interactions. By tuning the polarity of ELP sequences, we generate assemblies spanning micrometer- to centimeter-length scales that sediment within a few hours while preserving cellular metabolic activity. We demonstrate the portability of this platform across genetic backgrounds and inducible expression systems, and deploy it in an ethanologenic E. coli strain as a proof of principle. In small-scale fermentation settings, ELP-based ELMs enable controllable flocculation, reduce filtration time by more than threefold, and maintain ethanol production performance comparable to that of the parental strain. Together, this work establishes ELP surface display as a modular strategy for constructing de novo engineered living materials and defines initial genetic design rules linking molecular-scale interactions to emergent macroscopic organization.

synthetic biology↗

Harnessing CRISPR interference to re-sensitize laboratory strains and clinical isolates to last resort antibiotics

The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli. Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to test the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to >4-fold) and growth delay (up to 11-hours) in a medium-dependent fashion. To our knowledge, this is the first demonstration of CRISPRi-mediated re-sensitization to last-resort drugs in clinical isolates. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.

synthetic biology↗

The importance of cellulolytic and xylanolytic activities of Bacillus subtilis on dairy cow feed digestibility

A large body of literature has accumulated on the beneficial impact of the administration of Bacilli to dairy cows, particularly on dry matter intake, lactation performances and milk composition. In this work we sought to establish whether the ability of B. subtilis to secrete cellulase and xylanase enzymes could be involved in the positive effect exerted by these bacteria. Several feed ingredients were treated with two B. subtilis strains only differing for the amount of secreted cellulosolytic and xylanolytic enzymes, and feed quality was assessed. We found that in vitro fibre digestibility correlated with the activity of those enzymes. Our results revealed that B. subtilis cellulases and xylanases can effectively improve forage quality, providing a scientific rationale on the use of Bacilli as forage supplements to improve animal productivity. Moreover, a particular care was taken in designing a sustainable and economically viable Bacillus-based additive preparation process.

microbiology↗

dCas9 regulator to neutralize competition in CRISPRi circuits

CRISPRi-mediated gene repression allows simultaneous control of many genes. However, despite highly specific sgRNA-promoter binding, multiple sgRNAs still interfere with one another by competing for dCas9. We created a dCas9 regulator that adjusts dCas9 concentration based on sgRNAs demand, mitigating competition in CRISPRi-based logic gates. The regulators performance is demonstrated on both single-stage and layered CRISPRi logic gates and in two common E. coli strains. When a competitor sgRNA causes between two and ~25 fold-change in a logic gates input/output response without dCas9 regulator, the response is essentially unchanged when the regulator is used. The dCas9 regulator thus enables concurrent and independent operation of multiple sgRNAs, thereby supporting independent control of multiple genes.

synthetic biology↗

CRISPR interference as low burden logic inverters in synthetic circuits: characterization and tuning

The rational design of complex biological systems through the interconnection of single functional building blocks is hampered by many unpredictability sources; this is mainly due to the tangled context-dependency behavior of those parts once placed into an intrinsically complex living system. Among others, the finite amount of translational resources in prokaryotic cells leads to load effects in heterologous protein expression. As a result, hidden interactions among protein synthesis rates arise, leading to unexpected and counterintuitive behaviors. To face this issue in rational design of synthetic circuits in bacterial cells, CRISPR interference is here evaluated as genetic logic inverters with low translational resource usage, compared with traditional transcriptional regulators. This system has been studied and characterized in several circuit configurations. Each module composing the circuit architecture has been optimized in order to meet the desired specifications, and its reduced metabolic load has been eventually demonstrated via in-vivo assays.

synthetic biology↗