Biology subjects

Abil, Z.

Publications and source records attributed to Abil, Z..

3 recordsLinked to original sources

bioRxiv2026

CADGE 2.0, Transcription-Translation-Coupled DNA Replication is Improved in a Chemically Modified Cell-Free System

In vitro directed evolution in synthetic microcompartments can generally support the evolution of genes with functions beyond affinity. The main challenge in the implementation of this strategy is the need to incorporate no more than a single DNA template molecule per microcompartment, thereby establishing a robust genotype-phenotype linkage, but which results in slow, inconsistent in vitro transcription and translation (IVTT) and poor DNA recovery after selection or screening. To address this challenge, we previously developed CADGE (Clonal Amplification-enhanceD Gene Expression) a strategy that allows the clonal amplification of linear gene-encoding DNA and coupled, in situ transcription-translation of the gene of interest. Here, we show that clonal amplification is highly sensitive to the cell-free systems composition and that robust, highly efficient cell-free DNA amplification via the CADGE platform can be achieved by replacing standard vendor-supplied energy mixes with DNA replication-optimized, homemade counterparts.

synthetic biology↗

bioRxiv2024

Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template - encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage - expressed in the protein synthesis using recombinant elements (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system.

synthetic biology↗

bioRxiv2022

Clonal amplification-enhanced gene expression for cell-free directed evolution

In cell-free gene expression, low input DNA concentration severely limits the phenotypic output, which may impair in vitro protein evolution efforts. We address this challenge by developing CADGE, a strategy that is based on clonal isothermal amplification of a linear gene-encoding dsDNA template by the minimal {Phi}29 replication machinery and in situ transcription-translation. We demonstrate the utility of CADGE in bulk and in clonal liposome microcompartments to boost up the phenotypic output of soluble and membrane-associated proteins, as well as to facilitate the recovery of encapsulated DNA. Moreover, we report that CADGE enables the enrichment of a DNA variant from a mock gene library either via a positive feedback loop-based selection or high-throughput screening. This new biological tool can be implemented for cell-free protein engineering and the construction of a synthetic cell.

synthetic biology↗