Biology subjects

Golden, J. W.

Publications and source records attributed to Golden, J. W..

5 recordsLinked to original sources

bioRxiv2026

A set of constitutive promoters with graded strengths for gene expression in diverse cyanobacterial strains

Cyanobacteria have garnered interest as promising biological platforms for producing renewable biofuel, chemical feedstock, and bioactive molecules. For biotechnology applications, robust well-characterized genetic tools are required for genetically modifying cyanobacteria, but these tools are often developed for specific model strains. Here, we used broad host-range RSF1010-based plasmids to characterize a set of orthogonal constitutive promoters in diverse cyanobacterial strains. The promoters are random variants of the synthetic Escherichia coli PconII promoter. A library of PconII promoters driving a fluorescent reporter gene was first evaluated in Synechococcus elongatus and found to have a wide range of gene expression levels. A set of 25 promoter variants with graded strengths was selected after characterization in S. elongatus and three additional model cyanobacterial strains. To demonstrate the utility of these promoters, we isolated new genetically tractable cyanobacterial strains with high salt and alkalinity tolerance and transferred the subset of promoters into one of these newly isolated strains. Similar to the results with model strains, the subset of promoters had a wide range of expression levels in the non-model strain. These characterized promoters expand the genetic tools available for genetic engineering of model and non-model cyanobacterial strains. ImportanceThe use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications. Research AreasMicrobial genetics, plasmids and other genetic constructs, biotechnology Journal SecctionBiotechnology

microbiology↗

bioRxiv2025

Stability and characterization of infectious monkeypox virus extracellular virions in vitro2and vivo; implications for transmission, pathogenesis and treatment

Abstract/SummaryPoxviruses exist as multiple infectious morphogenic forms commonly simplified as mature virions (MV) and extracellular virions (EV). The roles of morphogenic subtypes as related to disease and transmission are enigmatic as EVs can exist both as cell associated (CEV) or released particles (rCEV) each with potentially unique biochemical properties impacting stability and infectivity. In vitro analysis of prototypical poxviruses is commonly utilized to infer larger conclusions about the in vivo function of all EV-like particles. Here we show that infectious EV of MPXV and VACV strains are stable for [≥] 14 weeks and are more sensitive to human or non-human primate complement compared to rabbit-derived complement. We also characterize the levels of EV produced during MPXV infection in NHPs and found temporal differences in production that may influence spread. Also, we present data characterizing and contrasting the EV from monkeypox (MPXV) and vaccinia virus (VACV) strains. Specifically, we quantified infectious EV quantities produced by different cultured cell lines and characterized the infectious properties and composition of the released (extracellular) virions. We conclude that A33 neutralizable cell associated-like virions (CEV-like), a form of EV, can significantly increase, depending on the strain of VACV or MPXV and the cell lines from which they were released. Based on the outcomes of our studies, the importance of understanding specific orthopoxvirus EV roles in a host-specific manner, as it relates to pathogenesis, stability, and transmission, is warranted and requires further study.

microbiology↗

bioRxiv2023

Phenotypically Complex Living Materials Containing Engineered Cyanobacteria

A cyanobacterial photosynthetic biocomposite material was fabricated using 3D-printing and bioengineered to produce multiple functional outputs in response to an external chemical stimulus. Our investigations show the advantages of utilizing additive manufacturing techniques in controlling the design and shape of the fabricated materials, which proved to be important for the support and growth of obligate phototrophic microorganisms within the material. As an initial proof-of-concept, a synthetic theophylline-responsive riboswitch in Synechococcus elongatus PCC 7942 was used for regulating the expression of a yellow fluorescent protein (YFP) reporter. Upon induction with theophylline, the encapsulated cells produced YFP within the hydrogel matrix. Subsequently, a strain of S. elongatus was engineered to produce an oxidative enzyme that is useful for bioremediation, laccase, expressed either constitutively or under the control of the riboswitch. The responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a useful tool in environmental bioremediation. Finally, cells were engineered to have the capacity for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing unintended environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in patterned volumetric 3D-printed designs, we demonstrate the potential of programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.

bioengineering↗

bioRxiv2020

Heterologous expression of cryptomaldamide in a cyanobacterial host

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, non-ribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drugs leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide, however high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/267179v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@660caforg.highwire.dtl.DTLVardef@1cab871org.highwire.dtl.DTLVardef@130de4org.highwire.dtl.DTLVardef@f50c64_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

The circadian clock and darkness control natural competence in cyanobacteria

Natural genetic competence-based transformation contributed to the evolution of prokaryotes, including the cyanobacterial phylum that established oxygenic photosynthesis. The cyanobacterium Synechococcus elongatus is noted both as a model system for analyzing a prokaryotic circadian clock and for its facile, but poorly understood, natural competence. Here a genome-wide screen aimed at determining the genetic basis of competence in cyanobacteria identified all genes required for natural transformation in S. elongatus, including conserved Type IV pilus, competence-associated, and newly described genes, and revealed that the circadian clock controls the process. The findings uncover a daily program that determines the state of competence in S. elongatus and adapts to seasonal changes of day-length. Pilus biogenesis occurs daily in the morning, but competence is maximal upon the coincidence of circadian dusk and the onset of darkness. As in heterotrophic bacteria, where natural competence is conditionally regulated by nutritional or other stress, cyanobacterial competence is conditional and is tied to the daily cycle set by the cells most critical nutritional source, the Sun.

microbiology↗