Nicholas Richard Sandoval

Harmful algal blooms (HABs) caused by Microcystis aeruginosa (MC) pose serious threats to freshwater ecosystems, public health, and water treatment infrastructure. Here, we present a targeted mitigation technology involving flocculation and settling of MC using a flocculant, polyaluminum chloride (PAC), and a nanotubular clay, halloysite, which serves both as a ballast to aid settling of the flocs and as a reservoir to deliver algaecides. The tubular morphology of halloysite allows loading of algaecide, thus constituting a targeted release of algaecide to MC cells in the floc. The specific algaecide chosen is a small-molecule polyphenol, propyl gallate (PG), whose release can be modulated by coating the halloysite nanotubes (HNT) with a thin layer of paraffin wax. Pulse amplitude modulation fluorometry (PAM) measurements confirm that PG disrupts the photosynthesis efficiency of MC in doses as low as 25 mg/L. Importantly, the extensive extracellular polymeric substance (EPS) produced by MC forms a hydrated and adhesive matrix that physically entraps HNTs within the floc through entanglement and interfacial interactions, thereby enhancing localized algaecide delivery while limiting exposure to nontarget organisms. Our findings demonstrate the potential of HNT-based delivery systems as scalable, environmentally benign technologies for managing freshwater HABs caused by Microcystis aeruginosa, with promising implications for long-term bloom suppression and ecological safety.

The commonly used arabinose- and rhamnose-inducible Escherichia coli promoters, PBAD and PRha, exhibit tight regulation through activation via their respective transcription factors, AraC and RhaS, alongside the cyclic AMP receptor protein. The mechanisms of these promoters have been characterized on a parts level, but nucleotide-level analysis has yet to be elucidated. Therefore, we describe here a massively parallel reporter assay that maps regulatory sites at the nucleotide level. The relative importance of nucleotides in each binding site is revealed, including loci not included in previous annotations. For PBAD, we confirm known sites and reveal novel binding sites involved in modulating gene expression. In PRha, we refine the length and sequence specificity of rhaI half-sites, updating previous annotations and providing nucleotide level insights into RhaS-mediated regulation. Mutations that lead to increased promoter strength, wider dynamic range, and altered basal expression are identified for both promoters. Engineered versions of PBAD and PRha promoters based on this data show improvements in dynamic range alongside a seven- and three-fold increase in promoter strength, respectively, with a slight increase in basal expression for the PBAD promoters and no significant increase for PRha. This work expands the genetic parts “toolkit” and increases the understanding of these important commonly used promoters.

Phage therapy to treat life-threatening drug-resistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of translation initiation factor IF-3 (infC) and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.

The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community.

Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ⁵⁴-dependent, butanol-responsive TF–promoter pair, BmoR-P_BMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P_BMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in P_BMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ⁵⁴-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.

Background: Ammonia concentrations typically increase during mammalian cell cultures, mainly due to glutamine and other amino acid consumption. An early ammonia stress indicator is a metabolic shift with respect to alanine. To determine the underlying mechanisms of this metabolic shift, a Chinese hamster ovary (CHO) cell line with two distinct ages (standard and young) was cultured in parallel fed‐batch bioreactors with 0 mM or 10 mM ammonia added at 12 h. Reduced viable cell densities were observed for the stressed cells, while viability was not significantly affected. The stressed cultures had higher alanine, lactate, and glutamate accumulation. Interestingly, the ammonia concentrations were similar by Day 8.5 for all cultures. We hypothesized the ammonia was converted to alanine as a coping mechanism. Interestingly, no significant differences were observed for metabolite profiles due to cell age. Glycosylation analysis showed the ammonia stress reduced galactosylation, sialylation, and fucosylation. Transcriptome analysis of the standard‐aged cultures indicated the ammonia stress had a limited impact on the transcriptome, where few of the significant changes were directly related metabolite or glycosylation reactions. These results indicate that mechanisms used to alleviate ammonia stress are most likely controlled post‐transcriptionally, and this is where future research should focus.

We report the ability to place a high concentration of liposomes in a confined volume as a multicompartment cluster that mimics biological cells and allows for the modulation of release of encapsulated species. The formation of these coated multicompartmental structures is achieved by first binding liposomes into clusters before encapsulating them within a two-dimensional metal organic framework composed of tannic acid coordinated with a metal ion. The essential feature is a molecularly thin skin over a system of clustered liposomes. The structural features of these pouches are revealed by small angle scattering and electron microscopy. Through cryogenic electron microscopy, clusters with intact liposomes are observed that appear to be encapsulated within a pouch. Small angle X-ray scattering shows the emergence of a relatively weak Bragg peak at q = 0.125Å^-1, possibly indicating the attachment of the bilayers of adjacent liposomes. The metal phenolic network (MPN) forms a nano-sized conformal coating around liposome clusters, resulting in the reduced release rate of the encapsulated rhodamine B dye. We further show the possibility of communication between the adjacent nano-compartments in the cluster by demonstrating enhanced energy transfer using Fluorescence Resonance Energy Transfer (FRET) experiments where the lipophilic donor dye 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) incorporated within one liposomal compartment transfers energy upon excitation to the lipophilic acceptor dye 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI) in a neighboring liposomal compartment due to their close proximity within the multicompartmental cluster. These observations have significance in adapting these multicompartmental structures that mimic biological cells for cascade reactions and as new depot drug delivery systems.