nanopore whole plasmid sequencing  (Plasmidsaurus)

Journal: bioRxiv

Article Title: Functional characterisation of an essential neo-chromosome III in Sc2.0 strain reveals opportunities and challenges for genome minimisation in Sc3.0

doi: 10.64898/2026.04.20.719597

Figure Lengend Snippet: A. Schematic diagram of the transcriptional unit (TU) for essential genes, consisting of a 500 bp promoter (p) upstream of the open reading frame (ORF), ORF, followed by a 200 bp terminator (t) downstream of the ORF, with a ROX site positioned downstream of the terminator. Although the serine tRNA gene SUP61 (tRNA-Ser) is an essential gene, it is not included in this design, as it will be incorporated into the tRNA neochromosome, which harbors all tRNA genes in the final synthetic strain. Each TU was PCR-amplified from genomic DNA of the wild-type (WT) strain BY4742, with 60 bp homology-flanking regions (HFRs) to adjacent TUs or the plasmid backbone. B. Schematic illustration of the construction strategy for assembling three essential mini-arrays via homologous recombination machinery (HR) in vivo in yeast. The first mini-array contains five essential genes (Egs) cloned into pRS413- HIS3 , the second contains five Egs in pRS415- LEU2 , and the third contains four Egs in pRS416- URA3 . All Egs are arranged unidirectionally and in a defined order, regardless of their native genomic orientation. Each mini-array is flanked by unique restriction enzyme sites designed to release the array in vitro . Each mini-array was assembled individually in vivo using the SynIII synthetic strain. Successful assembly was confirmed by junction PCR in combination with WT PCRTags. Following confirmation, the pRS plasmids harboring the essential mini-arrays were extracted using the EASY-c protocol and digested with restriction enzymes to release the essential mini-arrays. C. Schematic diagram illustrating the assembly of the three essential mini-arrays into a single construct via homologous recombination using pRS-KanMX6 as the destination vector in vivo in the SynIII strain. D. Successful assembly of eNeochrome III.V1 was confirmed using WT PCRTags, with the WT strain BY4742 serving as a positive control. E. eNeochrome III.V1 was extracted by EASY-c protocol and analysed by restriction digestion, revealing the expected digestion pattern. F. The EASY-c protocol was used to extract eNeochrome III.V1 from the SynIII strain, propagate it in bacteria, purify and linearize the eNeochrome III.V1 construct, and subject it to Nanopore sequencing. Nanopore sequencing analysis confirmed the correct assembly of eNeochrome III.V1.

Article Snippet: Whole-plasmid nanopore sequencing of selected constructs was additionally outsourced to Plasmidsaurus (UK).

Techniques: Amplification, Plasmid Preparation, Homologous Recombination, In Vivo, Clone Assay, In Vitro, Construct, Positive Control, Bacteria, Nanopore Sequencing

Journal: bioRxiv

Article Title: Functional characterisation of an essential neo-chromosome III in Sc2.0 strain reveals opportunities and challenges for genome minimisation in Sc3.0

doi: 10.64898/2026.04.20.719597

Figure Lengend Snippet: A. Each standardized biological part of the transcriptional unit (TU)—promoter (p), open reading frame (ORF), and terminator (t)—was PCR-amplified or synthesized with compatible overhangs and cloned into the HCKan vector using Golden Gate (GG) assembly to generate promoter library, ORF library and terminator library. B. Promoters of essential genes located on chromosome III from three yeast species ( S. cerevisiae , S. paradoxus , and S. eubayanus ) were individually cloned into HCKan-p vectors to generate three promoter libraries, each containing 14 promoters. The first library contains native promoters from S. cerevisiae , while the second and third libraries contain orthogonal promoters derived from S. paradoxus and S. eubayanus , respectively. ORFs corresponding to the essential genes on chromosome III were derived from wild-type S. cerevisiae BY4741 and cloned individually into HCKan-O vectors to generate a library of 14 ORFs. Terminators of essential genes on chromosome III were cloned into HCKan-t vectors to form three terminator libraries (14 terminators each): native S. cerevisiae terminators and orthogonal terminators from S. paradoxus and S. eubayanus . C. Native or orthogonal regulatory elements were designed to be compatible with ORF parts from different libraries. Individual TUs were assembled in a single one-pot (POT) Golden Gate reaction using POT- URA3 vectors. Newly constructed assemblies were verified by restriction digest analysis to release the insert using Bsa I. D. Two -three assembled essential Tus were sub-cloned into five YFASS vectors. The essential genes arranged in the same native order as in wild-type S. cerevisiae and correct assembly was verified by restriction digest analysis using Bsm BI. E Inserts were released and purified from the five YFASS vectors and flanked with 100-bp homologous flanking regions (HFRs) to facilitate in vivo assembly via homologous recombination (HR) in the SynIII strain to generate either linear or circular versions of eNeochrome III, using YAC12 as the destination vector. F. For linear constructs, the LEU2 marker located between telomeres was removed prior to final assembly. G. Correct assembly of eNeochrome III was confirmed by wild-type PCRTag analysis. Both linear (YAC12.L-eNeochrome.V3) and circular (YAC12.C-eNeochrome) forms were validated (for instance PCRTag analysis here is for third version of the eNeoIII.V3 were RE derive from S.paradoxus ). No product was observed for SynIII lacking eNeochrome III. H. Nanopore sequencing performed directly from yeast cells confirmed correct assembly of essential neochromosomes containing native or orthogonal regulatory elements for eNeochrome III variants (V2, V3, and V4). I . Linear constructs yielded substantially higher read coverage compared to circular constructs assembled with the same regulatory configurations.

Article Snippet: Whole-plasmid nanopore sequencing of selected constructs was additionally outsourced to Plasmidsaurus (UK).

Techniques: Amplification, Synthesized, Clone Assay, Plasmid Preparation, Derivative Assay, Construct, Purification, In Vivo, Homologous Recombination, Marker, Nanopore Sequencing

Journal: bioRxiv

Article Title: Functional characterisation of an essential neo-chromosome III in Sc2.0 strain reveals opportunities and challenges for genome minimisation in Sc3.0

doi: 10.64898/2026.04.20.719597

Figure Lengend Snippet: A. Comprehensive phenotypic analysis of SynIII strains harboring eNeochrome III.v1. Ten-fold serial dilutions were prepared from overnight cultures of SynIII strains carrying the neo-essential chromosome III, starting from an OD₆₀₀ of 0.1. Strains were identified by their corresponding strain IDs listed on the left. Spot assays were performed for SynIII strains harboring eNeochrome III.v1 on a pRS vector. Control strains carrying the empty pRS-KanMX6 vector (WT BY4742 + pRS-KanMX6 and SynIII + pRS-KanMX6) were included for comparison. Spot tests were conducted on selective YPD + G418 media at 25°C, 30°C, and 37°C, as well as under multiple stress conditions, including SM + camptothecin (5µg/ml), YPEG (2% glycerol + 2% ethanol), SM + 6-azauracil (100µg/ml), SM + benomyl (5µg/ml), SM + hydroxyurea (0.2M), YPD + MMS (0. 05%), SM + cycloheximide (10 μg/mL, 2 h pre-treatment), and YPD + H₂O₂ (1 mM, 2 h pre-treatment). Additional assays were performed on SM supplemented with sorbitol (2M), low pH (pH 4.0), and high pH (pH 9.0). The first-generation SynIII + pRS–eNeochrome III.v1 strain (1g), where g1 denotes the first generation of the constructed strain, exhibited a pronounced growth defect under most tested conditions. Notably, growth fitness was restored after prolonged passaging, as observed in the 100-generation strain (100g). Unexpectedly, the SynIII + pRS–eNeochrome III.v1-strain (1g) displayed similar growth to controls when exposed to YPD + G418 supplemented with camptothecin or 6-azauracil. Both SynIII + pRS–eNeochrome III.v1 strains (1g and 100g) failed to grow on media containing non-fermentable carbon sources as the sole energy source. Nanopore sequencing revealed loss of mitochondrial DNA in these strains following introduction of pRS–eNeochrome III.v1 into the semi-synthetic SynIII background. B. Comprehensive phenotypic analysis of SynIII strains harboring eNeochrome III.v2 S.c , III.v3 S.p , and III.v4 S.e engineered with native or orthogonal recombination elements (REs) derived from S. cerevisiae , S. paradoxus , and S. eubayanus on the YAC12 vector. Control strains carrying the empty vector with HIS3 -marker (SynIII+pRS413 and WT BY4742+pRS413) were included for comparison. Ten-fold serial dilutions were prepared from overnight cultures of SynIII strains carrying different versions of the neo-essential chromosome III, starting from an OD₆₀₀ of 0.1. Strains were identified by their corresponding strain IDs listed on the left. Spot assays were performed on SC–His selective media and imaged after three days of incubation. Most SynIII strains harboring essential neochromosomes on YAC12 exhibited robust growth across tested conditions. However, a severe growth defect was observed in SynIII+YAC12.C– eNeochrome III.v2 ( S. cerevisiae ) and a moderate defect in SynIII+YAC12.L–eNeochrome III.v4 ( S. eubayanus ) upon camptothecin treatment. In contrast, SynIII+YAC12.C–eNeochrome III.v3 ( S. paradoxus ) displayed pronounced resistance to camptothecin. Both SynIII+pRS413 and SynIII+YAC12.C– eNeochrome III.v2 ( S. cerevisiae ) exhibited sensitivity to MMS, whereas SynIII+YAC12.L–eNeochrome III.v2 ( S. cerevisiae ) and SynIII+YAC12.C–eNeochrome III.v3 ( S. paradoxus ) showed resistance comparable to WT BY4742 + pRS413. Notably, strains harboring essential neo chromosomes with recombination elements derived from S. cerevisiae exhibited marked growth impairment at high pH (pH 9.0) compared with SynIII strains carrying refactored recombination elements. Although the underlying mechanism remains unclear, we anticipate that future transcriptomic and proteomic analyses will provide mechanistic insight. (move to the text)

Article Snippet: Whole-plasmid nanopore sequencing of selected constructs was additionally outsourced to Plasmidsaurus (UK).

Techniques: Plasmid Preparation, Control, Comparison, Construct, Passaging, Nanopore Sequencing, Derivative Assay, Marker, Incubation

Journal: bioRxiv

Article Title: Functional characterisation of an essential neo-chromosome III in Sc2.0 strain reveals opportunities and challenges for genome minimisation in Sc3.0

doi: 10.64898/2026.04.20.719597

Figure Lengend Snippet: A. Diagram of the SCRaMbLE reporter ERICA (Elementary Random Integration Cassette). The ERICA cassette contains a URA3 expression cassette (promoter–ORF–terminator) flanked by 34 bp loxPsym sites. These loxPsym sites enable random integration into any compatible loxPsym site within the synthetic chromosome via homologous recombination, eliminating the need for locus-specific oligo design. B . Schematic overview of the workflow used to minimize synthetic chromosome size. SynIII+pRS-eNeo.V1 S. cerevisiae serves as the starter strain. ERICA is randomly integrated into loxPsym sites on synthetic chromosome III to generate a SynIII strain harboring eNeoChromIII.V1, which is used as a pooled population. SCRaMbLE is induced for 3, 5, 7, 24, or 72 hours using Cre recombinase driven by either the daughter-specific promoter pSCW11 or the strong constitutive TDH3 promoter. Post-SCRaMbLE mutants that have lost the reporter are positively selected on 5-FOA medium. PCRTag analysis of genomic DNA is used to estimate gene deletions, and nanopore sequencing resolves structural variation, including deletions, duplications, inversions, and insertions. These data reveal deletion patterns and identify SCRaMbLE hot and cold spots. Mutants carrying the highest deletion burden are subjected to sequential rounds of SCRaMbLE. C. Analysing post-SCRaMbLEd mutants harboring minimal genomes using nanopore technology

Article Snippet: Whole-plasmid nanopore sequencing of selected constructs was additionally outsourced to Plasmidsaurus (UK).

Techniques: Expressing, Homologous Recombination, Nanopore Sequencing