Biology subjects

Malyshava, A.

Publications and source records attributed to Malyshava, A..

2 recordsLinked to original sources

bioRxiv2026

Synthetic Combinatorial Minimisation of Cell Cycle Control in Yeast

The eukaryotic cell cycle, with its inherent regulatory redundancy, provides an ideal target for exploring genome modularisation and minimisation through synthetic genomics. Building upon principles established by the Synthetic Yeast Genome (Sc2.0) project, we used CRISPR-mediated genome engineering to relocate nine key cell cycle genes into a synthetic gene cluster in the S. cerevisiae genome, to allow combinatorial study of these genes in yeast. We employed Cre/loxP recombination to rapidly generate hundreds of strains with different gene deletion combinations in the module, with the objective of identifying minimal gene sets that permit robust cell cycle function. Using FACS-sorting and POLAR (Pool of Long Amplified Reads) sequencing, we conducted high-throughput analysis of gene deletion combinations in large cell pools, detecting approximately 80% of theoretically possible gene combinations, including those predicted from prior mathematical modelling studies. Our findings demonstrate that the cell cycle gene set can be minimised while maintaining viability, though only select combinations of gene deletions ensure robust fitness across different conditions. This work establishes a framework for genome minimisation, opening the door to the design of simplified, modular synthetic genomes for diverse applications.

synthetic biology↗

bioRxiv2024

Combinatorial Design Testing in Genomes with POLAR-seq

Synthetic biology projects increasingly use modular DNA assembly or synthetic in vivo recombination to generate diverse combinatorial libraries of genetic constructs for testing. But as these designs expand to multigene systems it becomes challenging to sequence these in a cost-effective way that reveals the genotype to phenotype relationships in the libraries. Here, we introduce a new quick, low-cost method designed for assessing combinational designs of genome-integrated multigene constructs that we call Pool of Long Amplified Reads (POLAR) sequencing. POLAR-seq takes genomic DNA isolated from library pools and uses long range PCR to amplify target genomic regions up to 35 kb long containing combinatorial designs. The pool of long amplicons is then directly read by nanopore sequencing with full length reads then used to identify the gene content and structural variation of individual genotypes in the library and read count indicating how abundant a genotype is within the pool. Using yeast cells with loxP-containing synthetic gene clusters that rearrange in vivo in the presence of Cre recombinase, we demonstrate how POLAR-seq can be used to identify global patterns from combinatorial experiments, find the most abundant genotypes in a pool and also be adapted to sequence-verify gene clusters from isolated strains.

synthetic biology↗