Biology subjects

Levrier, A.

Publications and source records attributed to Levrier, A..

4 recordsLinked to original sources

bioRxiv2025

A synthetic cell phage cycle

Viral infection of living cells, exemplified by bacteriophage interaction with bacteria, is fundamental to biology and universal across living systems. Here, we establish an all-cell-free viral cycle where T7 phages infect synthetic cells, equipped with lipopolysaccharides on the outer leaflet of the lipid membrane while encapsulating a cell-free gene expression system. We track each cycle step to demonstrate T7 phage-specific adsorption onto the liposomes, genome ejection, replication, expression, and assembly of new infectious virions within the synthetic cells. We quantify key characteristics of the cycle, including the multiplicity of infection, replication efficiency, liposome size constraints, and phage rebinding dynamics. This work establishes a versatile, fully defined in vitro platform for reconstructing and investigating viral infections from individual molecular components.

synthetic biology↗

bioRxiv2024

Split minireporters facilitate monitoring of gene expression and peptide production in linear cell-free transcription-translation systems

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present minireporters based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that minireporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP, and matching with in vivo expression measurements. We also use minireporters as small fusion tags to measure TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent minireporter based on split-nanoluciferase. The [~]80% gene sequence length reduction afforded by minireporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of minireporters compared to full-length GFP is less likely to bias peptide solubility assays. We anticipate that minireporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

synthetic biology↗

bioRxiv2023

PHEIGES, all-cell-free phage synthesis and selection from engineered genomes

Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.

synthetic biology↗

bioRxiv2021

Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from {sigma}70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the {Delta}recBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

synthetic biology↗