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Karas, B. J.

Publications and source records attributed to Karas, B. J..

At least 19 recordsLinked to original sources

Clinical Campylobacter jejuni isolates: genomes and genetic tools

Campylobacter jejuni is a major cause of food-borne gastroenteritis and is responsible for substantial mortality and economic losses in meat and dairy production. Detecting C. jejuni in contaminated food samples remains difficult because current assays are culture-based, slow, and can yield false positives. As a result, contamination may not be identified for several days, limiting detection at the point of production. Developing improved assays has also been challenging because Campylobacter genetics and the biology of clinical isolates remain poorly understood. Here, we expand the C. jejuni genetic toolbox by sequencing two strains, HC1 and RM1164, derived from patient and food samples. We identified two cryptic plasmids in HC1, one potentially capable of conjugation and another conferring tetracycline resistance. We also engineered a mobilizable plasmid carrying an OriT sequence that can be transferred from Escherichia coli donor strains to C. jejuni RM1164 by conjugation. Together, these clinical isolates and the plasmid system expand the genetic tools available for C. jejuni.

synthetic biology↗

Conjugation Based CRISPR Antifungals (COBRA)

Fungal infections are increasingly difficult to treat due to rising antifungal resistance and the limited number of effective drug classes. To address this challenge, we developed a conjugation-based CRISPR antifungal (COBRA) platform that enables delivery of programmable gene-targeting machinery from Escherichia coli to Saccharomyces cerevisiae. We engineered a mobilizable pVenom plasmids containing an oriT for conjugation, Cas9 and guide RNAs for elimination of yeast. To prevent toxicity in E. coli, yeast ACT1 intron is inserted in Cas9. Seven guide RNAs targeting essential genes involved in cell cycle progression, ribosome function, and DNA replication were first assessed by using electroporation as delivery and demonstrated strong lethality for guides targeting CDC28 and MCM2. When delivered by conjugation, these guides again reproduced these results, whereas an intron-targeting control remained non-lethal. Dual-guide plasmids eliminated yeast colony formation entirely. Together, these findings demonstrate that conjugation-delivered CRISPR machinery can successfully target endogenous fungal genes and, especially when multiplexed, offers an effective and programmable antifungal strategy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/692781v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1fce050org.highwire.dtl.DTLVardef@ead257org.highwire.dtl.DTLVardef@978391org.highwire.dtl.DTLVardef@13ead19_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Conjugation-mediated DNA delivery to the filamentous fungus Ustilago maydis

Phytopathogenic fungi are ubiquitous throughout the environment and threaten global food security. This issue is further amplified by the increasing resistance of pathogens to antimicrobials. Current chemical-based antifungals target cells by inhibiting growth or metabolic function, making them ideal for fungal gain of resistance mutations. Biofungicides are a rising class of antifungals that have low potential for negative environmental impact and provide the fungi almost no potential for gaining resistance. Conjugative plasmids which play a role in the natural mechanism of horizontal gene transfer in bacteria, have been repurposed to deliver toxic genetic cargo to recipient cells, showing promise as next-generation antimicrobial agents. In this work, we have demonstrated the first protocol for delivering DNA from Escherichia coli to the filamentous phytopathogen, Ustilago maydis through conjugation. DNA delivery was confirmed using PCR screening of DNA isolated from the re-streaked transconjugants. Although challenges such as reduced conjugation efficiency and extrachromosomal replication persist, this work establishes the first step towards creating a conjugation-based biofungicide.

synthetic biology↗

Resolving replication incompatibility between chloroplast and conjugative plasmids in E. coli

Chloroplast genomes present a promising chassis for engineering photosynthetic eukaryotes, but efficient delivery of large DNA constructs back into the organelle remains a major technical barrier. Conventional transformation methods rely on purified DNA and physical force to drive uptake into the chloroplast, often resulting in DNA shearing and thus low transfer efficiency for large constructs. Bacterial conjugation offers an attractive alternative as this is an entirely in vivo process, enabling DNA transfer without any physical manipulation. To assess the feasibility of this approach, we attempted to generate an Escherichia coli donor strain carrying both the broad-host-range conjugative plasmid pTA-Mob (52.7 kb) and the cloned Phaeodactylum tricornutum chloroplast genome (pPt_Cp, 132.9 kb). Unexpectedly, pTA-Mob and pPt_Cp proved incompatible: co-maintenance could not be achieved via electroporation, and conjugation with a self-transmissible pTA-Mob variant resulted in a [~]10{square}-fold decrease in transfer efficiency. Systematic testing of pTA-Mob and pPt_Cp plasmid variants as well as sequence analysis of evolved transconjugants revealed that the incompatibility arose from the pBBR1 replicon present in pTA-Mob. Guided by these insights, we identified an alternative conjugative plasmid, pRL443, that was compatible with pPt_Cp. Together, these findings provide a framework for dissecting plasmid incompatibility when working with large constructs and establish a functional conjugative system capable of mobilizing the P. tricornutum chloroplast genome.

synthetic biology↗

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↗

Engineering Conjugative Plasmids for Inducible Horizontal DNA Transfer

Rapidly developing microbial resistance to existing antimicrobials poses a growing threat to public health and global food security. Current chemical-based treatments target cells by inhibiting growth or metabolic function, but their effectiveness is diminishing. To address the growing antimicrobial resistance crisis, there is an urgent need for innovative therapies. Conjugative plasmids, a natural mechanism of horizontal gene transfer in bacteria, have been repurposed to deliver toxic genetic cargo to recipient cells, showing promise as next-generation antimicrobial agents. However, the ecological risks posed by unintended gene transfer require robust biocontainment strategies. In this study, we developed inducible conjugative plasmids to solve these challenges. Utilizing an arabinose-inducible promoter, we evaluated 13 plasmids with single essential gene deletions, identifying trbC and trbF as strong candidates for stringent regulation. These plasmids demonstrated inducibility in both cis and trans configurations, with induction resulting in up to a 5-log increase in conjugation efficiency compared to uninduced conditions. Although challenges such as reduced conjugation efficiency and promoter leakiness persist, this work establishes a foundation for the controlled transfer of plasmids, paving the way for safer and more effective antimicrobial technologies.

synthetic biology↗

Spheroplasted cells: a game changer for DNA delivery to diatoms

Diatoms produce 20% of the worlds fixed organic carbon annually, making them vital to global carbon fixation and climate change mitigation. Their potential as cell factories for biofuels, proteins, and other high value chemicals remains underutilized due to a lack of genetic engineering tools, with DNA delivery being one of the biggest challenges. Here, we present optimized electroporation and polyethylene glycol transformation methods for delivering DNA and ribonucleoprotein complexes to Phaeodactylum tricornutum, a model diatom species and emerging chassis for algal biotechnology. It was possible to recover transformants with as little as 1 ng of DNA, and to transform linear or circular episomes as large as 55.6 kb. With the optimized electroporation protocol, episomes can be assembled in the algal cell de novo through diatom in vivo assembly (DIVA), forgoing the need for time-consuming traditional cloning steps in Escherichia coli and Saccharomyces cerevisiae. It was also possible to electroporate a Cas9 ribonucleoprotein complex in P. tricornutum, providing an alternative to biolistics for DNA free genome engineering. We have demonstrated that the PEG approach can be adapted to successfully transform Thalassiosira pseudonana, demonstrating the applicability of our methods for engineering other diatom species. These tools can be used to accelerate diatom synthetic biology projects and, therefore, the development of sustainable technologies.

synthetic biology↗

PEG Adjustment Enables Genome Transplantation Using Mycoplasma Mycoides Recipient

Pioneering advances in synthetic biology, initiated at the J. Craig Venter Institute, have enabled the creation of the first cell driven by synthetic genome and opened a new era of creating designer microbes. This technology offers far greater potential for genetic modification than traditional genome editing techniques and holds promise for the routine creation of synthetic cells as DNA synthesis costs decrease and genetic knowledge expands. Two essential technologies underpin this achievement: the ability to clone entire genomes in host organisms, such as Saccharomyces cerevisiae, and the successful transplant of these genomes into recipient cells to generate living organisms. In all previous work the recipient cell in genome transplantation experiments has been Mycoplasma capricolum. In this study, we explored the potential of using Mycoplasma mycoides strains as recipient cells for genome transplantation. By increasing polyethylene glycol (PEG) concentration from 5% to 10%, we successfully transplanted various M. mycoides strains using several M. mycoides recipient cells. Additionally, we demonstrated the ability to transplant M. capricolum genomes into M. mycoides recipient cells; although with lower efficiency compared to M. mycoides strains. These findings provide a modified transplantation protocol and likely get us closer to expanding genome transplantation to other bacterial species.

synthetic biology↗

Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation to Haemophilus influenzae Rd

Haemophilus influenzae is a bacterial species of interest for its medical relevance and utility as a model system. Despite its role in several landmark molecular and synthetic biology studies, H. influenzae remains underexplored as a potential chassis organism. The limited availability of reliable and convenient transformation methods and genetic tools for H. influenzae are obstacles to this end. However, a strain of H. influenzae Rd KW20 lacking the type II restriction endonucleases HindII and HindIII has previously been developed. Here, we show that this strain is more readily transformable by electroporation than wild-type Rd KW20. We also developed a series of multi-host plasmids carrying antibiotic selection and fluorescent visual markers based on the pSU20 vector. The availability of H. influenzae {Delta}HindII/III, paired with the electroporation method and plasmids presented here, will promote the exploration of H. influenzae as a host organism for synthetic biology applications.

synthetic biology↗

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonium, they have a great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have improved genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants or symbiotic association with non-legumes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/572671v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@70e377org.highwire.dtl.DTLVardef@165b06corg.highwire.dtl.DTLVardef@12196a9org.highwire.dtl.DTLVardef@2a33e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Design and assembly of the 117-kb Phaeodactylum tricornutum chloroplast genome

There is a growing impetus to expand the repository of chassis available to synthetic biologists. The chloroplast genome presents a unique chassis for engineering photosynthetic eukaryotes by virtue of its compact size, lack of epigenetic regulation, and containment within the secluded lipid bilayers of the organelle. The development of the chloroplast as a synthetic biology chassis, however, has been limited by a lack of efficient techniques for whole genome cloning and engineering. Here, we demonstrate two approaches for cloning the 117 kb Phaeodactylum tricornutum chloroplast genome that have 90 to 100% efficiency when screening as few as ten Saccharomyces cerevisiae colonies following yeast assembly. The first method directly uses PCR-amplified fragments of the genome for yeast assembly, whereas the second method relies upon the pre-cloning of eight overlapping genomic regions into individual plasmids that they can later be released from. The cloned genome can be stably maintained and propagated within Escherichia coli, which provides an exciting opportunity for engineering a novel delivery mechanism for bringing DNA directly to the algal chloroplast. As well, one of the cloned genomes was designed to contain a single SapI site within the yeast URA3 open-reading frame, which can be used to linearize the genome and integrate designer cassettes via golden-gate cloning or further iterations of yeast assembly.

synthetic biology↗

De novo synthesis of a conjugative system from human gut metagenomic data for targeted delivery of Cas9 antimicrobials

Metagenomic sequence represents an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequence requires the identification of suitable candidate systems as starting scaffolds for de novo DNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic trash bin for conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines. Using a human metagenomic gut dataset representing 2805 taxonomically distinct units, we identified 1598 contigs containing conjugative systems with a differential distribution in human cohorts. We synthesized de novo an entire Citrobacter spp. conjugative system of 54 kb and containing at least 47 genes, pCitro, and found that pCitro conjugates from Escherichia coli to Citrobacter rodentium with a 30-fold higher frequency than to E. coli, and is compatible with Citrobacter resident plasmids. Mutations in the traV and traY conjugation components of pCitro inhibited conjugation. We showed that pCitro can be re-purposed as an antimicrobial delivery agent by programming it with the TevCas9 nuclease and Citrobacter -specific sgRNAs to kill C. rodentium. Our study reveals a trove of uncharacterized conjugative systems in metagenomic data and describes an experimental framework to animate these large genetic systems as novel target-adapted delivery vectors for Cas9-based editing of bacterial genomes.

synthetic biology↗

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↗

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↗

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↗

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↗

Genetic requirements for cell division in a genomically minimal cell

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. While this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics resulting in irregular morphologies. The addition of 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents significantly less morphological variation than JCVI-syn3.0. We further identified seven of these 19 genes, including two known cell division genes ftsZ and sepF and five genes of unknown function, required together to restore cell morphology and division similar to JCVI-syn1.0. This surprising result emphasizes the polygenic nature of cell morphology, as well as the importance of a Z-ring and membrane properties in the physiology of genomically minimal cells.

synthetic biology↗

Cas9-generated auxotrophs of Phaeodactylum tricornutum are characterized by small and large deletions that can be complemented by plasmid-based genes

The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ~2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

synthetic biology↗