Biology subjects

Giguere, D. J.

Publications and source records attributed to Giguere, D. J..

4 recordsLinked to original sources

bioRxiv2022

Towards the creation of Acholeplasma laidlawii driven by synthetic genomes

Assembling synthetic bacterial genomes in yeast and genome transplantation has enabled an unmatched level of bacterial strain engineering, giving rise to cells with minimal and chemically synthetic genomes. However, this technology is currently limited to members of the Spiroplasma phylogenetic group, mostly Mycoplasmas, within the Mollicute class. Here, we propose new genetic tools for developing these technologies for Acholeplasma laidlawii, which is phylogenetically distant from Mycoplasmas and, unlike most Mollicutes, uses a standard genetic code. We first investigated a donor-recipient relationship between two A. laidlawii strains through whole-genome sequencing. We then created multi-host shuttle plasmids and used them to optimize an electroporation protocol. We also demonstrated the use of evolution to create superior strains for DNA uptake via electroporation. For genome transplantation, we selected A. laidlawii 8195 as the recipient strain and created a PG-8A donor strain by inserting a Tn5 transposon carrying a tetracycline resistance gene. The tools presented here will improve Acholeplasma research and accelerate the effort toward creating A. laidlawii strains driven by synthetic genomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/508766v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1d48427org.highwire.dtl.DTLVardef@13056d3org.highwire.dtl.DTLVardef@9452f3org.highwire.dtl.DTLVardef@1d1f3b8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Conjugation-based genome engineering in Deinococcus radiodurans

D. radiodurans has become an attractive microbial platform for the study of extremophile biology and industrial bioproduction. To improve the genomic manipulation and tractability of this species, the development of tools for whole genome engineering and design is necessary. Here, we report the development of a simple and robust conjugation-based transformation system from E. coli to D. radiodurans allowing for the introduction of stable, replicating plasmids expressing antibiotic resistance markers. Using this method with nonreplicating plasmids, we developed a protocol for creating sequential gene deletions in D. radiodurans by targeting re-striction-modification system genes. Importantly, we demonstrated a conjugation-based method for cloning the large (178 kb), high G+C content MP1 megaplasmid from D. radiodurans in E. coli. The conjugation-based tools described here will facilitate the development of D. radiodurans strains with synthetic genomes for biological studies and industrial applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/464295v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@128437borg.highwire.dtl.DTLVardef@6669ddorg.highwire.dtl.DTLVardef@1b041eaorg.highwire.dtl.DTLVardef@b77f07_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Telomere-to-telomere genome assembly of Phaeodactylum tricornutum

Phaeodactylum tricornutum is a marine diatom with a growing genetic toolbox available and is being used in many synthetic biology applications. While most of the genome has been assembled, the currently available genome assembly is not a completed telomere-to-telomere assembly. Here, we used Oxford Nanopore long reads to build a telomere-to-telomere genome for Phaeodactylum tricornutum. We developed a graph-based approach to extract all unique telomeres, and used this information to manually correct assembly errors. In total, we found 25 nuclear chromosomes that comprise all previously assembled fragments, in addition to the chloroplast and mitochondrial genomes. We found that chromosome 19 has filtered long-read coverage and a quality estimate that suggests significantly less haplotype sequence variation than the other chromosomes. This work improves upon the previous genome assembly and provides new opportunities for genetic engineering of this species, including creating designer synthetic chromosomes.

genomics↗

bioRxiv2020

Complete and validated genomes from a metagenome

0.1The assembly and binning of metagenomically-assembled genomes (MAGs) using Illumina sequencing has improved the genomic characterization of unculturable communities. However, short-read-only metagenomic assemblies rarely result in completed genomes because of the difficulty assembling repetitive regions. Here, we present a strategy to complete and validate multiple MAGs from a bacterial community using a combination of short and ultra long reads (N50 > 25 kb). Our strategy is to perform an initial long read-only metagenomic assembly using metaFlye, followed by multiple rounds of polishing using both long and short reads. To validate the genomes, we verified that longs reads spanned the regions that were not supported by uniquely mapped paired-end Illumina sequences. We obtained multiple complete genomes from a naphthenic acid-degrading community, including one from the recently proposed Candidate Phyla Radiation. The majority of the population is represented by the assembled genomes; recruiting 63.77 % of Nanopore reads, and 64.38 % of Illumina reads. The pipeline we developed will enable researchers to validate genomes from metagenomic assemblies, increasing the quality of metagenomically assembled genomes through additional scrutiny.

bioinformatics↗