Biology subjects

Ciurkot, K.

Publications and source records attributed to Ciurkot, K..

4 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

Iterative SCRaMbLE for Engineering Synthetic Genome Modules and Chromosomes

Synthetic biology offers the possibility of synthetic genomes with customised gene content and modular organisation. In eukaryotes, building whole custom genomes is still many years away, but work in Saccharomyces cerevisiae yeast is closing-in on the first synthetic eukaryotic genome with genome-wide design changes. A key design change throughout the synthetic yeast genome is the introduction of LoxPsym site sequences. These enable inducible genomic rearrangements in vivo via expression of Cre recombinase via SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution). When paired with selection, SCRaMbLE can quickly generate strains with phenotype improvements by diversifying gene arrangement and content in LoxPsym-containing regions. Here, we demonstrate how iterative cycles of SCRaMbLE can be used to reorganise synthetic genome modules and synthetic chromosomes for improved functional performance under selection. To achieve this, we developed SCOUT (SCRaMbLE Continuous Output and Universal Tracker), a reporter system that allows SCRaMbLEd cells to be sorted into a high diversity pool. When coupled with long-read sequencing, SCOUT enables high-throughput mapping of genotype abundance and correlation of gene content and arrangement with growth-related phenotypes. Iterative SCRaMbLE was applied here to yeast strains with a full synthetic chromosome, and to strains with synthetic genome modules encoding the gene set for histidine biosynthesis. Five synthetic designs for HIS modules were constructed and tested, and we investigated how SCRaMbLE reorganised the poorest performing design to give improved growth under selection. The results of iterative SCRaMbLE serve as a quick route to identify genome module designs with optimised function in a selected condition and offer a powerful tool to generate datasets that can inform the design of modular genomes in the future.

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↗

bioRxiv2022

Synthetic yeast chromosome XI design enables extrachromosomal circular DNA formation on demand

We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.

synthetic biology↗