Biology subjects

Nucifora, D. P.

Publications and source records attributed to Nucifora, D. P..

3 recordsLinked to original sources

bioRxiv2025

Direct Genome Transfer from Acholeplasma laidlawii to Yeast

Cloning bacterial genomes in yeast or other hosts is a crucial step in workflows for creating highly engineered strains. The genome of Acholeplasma laidlawii can be cloned in yeast if a toxic gene is removed, but this deletion has not been tested in live bacteria. Here, we demonstrate cloning the 1.46 Mb genome of A. laidlawii strain DN-E by integrating a yeast vector into the toxic gene and transferring the genome directly to yeast using cell fusion. Genomic integration was only possible when A. laidlawii was complemented with a functional recA gene. Following cell fusion, 13 out of 20 screened yeast colonies contained the A. laidlawii genome, as determined by multiplex PCR. This is the first demonstration showing cloning entire A. laidlawii genomes by simply mixing donor and recipient cells in fusion buffer, bringing us one step closer to creating fully synthetic Acholeplasma strains. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/683135v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1429a68org.highwire.dtl.DTLVardef@1a754c1org.highwire.dtl.DTLVardef@ad6044org.highwire.dtl.DTLVardef@8b2539_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

SLICER: Seamless Loss of Integrated Cassettes Using Endonuclease Cleavage and Recombination in Deinococcus radiodurans

Methods for creating seamless genome modifications are an essential part of the microbial genetic toolkit that allows for strain engineering through the recycling of selectable markers. Here, we report the development of a method, termed SLICER, which can be used to create seamless genome modifications in D. radiodurans. We used SLICER to sequentially target four putative restriction-modification (R-M) system genes, recycling the same selective and screening markers for each subsequent deletion. A fifth R-M gene was replaced by a selectable marker to create a final D. radiodurans strain with 5 of the 6 putative R-M systems deleted. While we observed no significant increase in transformation efficiency, SLICER is a promising method to obtain a fully restriction-minus strain and expand the synthetic biology applications of D. radiodurans including as an in vivo DNA assembly platform. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/512367v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@13a4f39org.highwire.dtl.DTLVardef@127655corg.highwire.dtl.DTLVardef@16faddforg.highwire.dtl.DTLVardef@10023cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

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↗