Biology subjects

Kelly, C. L.

Publications and source records attributed to Kelly, C. L..

7 recordsLinked to original sources

bioRxiv2026

Identification of a native Type I Secretion System cargo in Zymomonas mobilis and its application for extracellular enzyme secretion

Engineering the ethanologenic Gram-negative bacterium Z. mobilis for the secretion of hydrolytic enzymes is a key step towards establishing a biofuel cell factory that uses complex waste material as feedstock. Secretion strategies in Z. mobilis have exclusively relied on signal peptides, which limit protein transport to the periplasm. To achieve single-step secretion across the Z. mobilis double-layered membrane, we sought to identify a native Type I Secretion System (T1SS) tag for fusion to proteins of interest. While a T1SS operon had been identified in the Z. mobilis genome, its native cargo had remained unknown and the use of T1SS secretion tags had so far been unexplored. Here, bioinformatic analysis identified the Major Intrinsic Protein (MIP) as a putative T1SS cargo, and its role validated through fusion of C-terminal sequences of two lengths (61 and 141 amino acids) to a heterologous {beta}-galactosidase from Bacteroides thetaiotaomicron, expressed in Z. mobilis. The 141 amino acid tag, including two RTX domains, resulted in significantly higher secretion efficiency than the 61 amino acid tag lacking RTX repeats, consistent with the established role of RTX domains in preventing premature cytoplasmic folding, thus improving secretion. As extracellular secretion of hydrolytic enzymes has remained a major bottleneck in the development of Z. mobilis as a sustainable cell factory, the identification of a native T1SS secretion tag directly addresses this limitation, introducing a novel tool for enzyme delivery.

synthetic biology↗

bioRxiv2025

A comprehensive genetic toolkit for Zymomonas mobilis; allowing rapid, high-resolution, combinatorial engineering of metabolic pathways.

The limited availability of reliable genetic tools has constrained the precise engineering of Zymomonas mobilis, a promising bacterium for the sustainable bioproduction of fuels and commodity chemicals. Here, we present a comprehensive, modular genetic toolkit that enables high-resolution, combinatorial control of gene expression in Z. mobilis. We constructed and characterised a synthetic promoter library spanning the broadest dynamic range reported for this organism, alongside a suite of strong transcriptional terminators that effectively insulate genetic cassettes and prevent transcriptional readthrough. To achieve predictable translation across diverse genetic contexts, we further implemented bicistronic design (BCD) elements, allowing context-independent control of expression. Promoter activities were shown to be stable under both aerobic and anaerobic conditions, highlighting the robustness of the regulatory parts across physiologically relevant environments. The toolkit was integrated into the Start-Stop Assembly system, facilitating high-throughput construction of multi-gene pathways. We demonstrate its utility through the rapid assembly and testing of the possible expression space of genes encoding a 2,3-butanediol biosynthetic pathway. Collectively, this genetic toolkit now allows the high-resolution engineering of multi-enzyme pathways in Z. mobilis for industrial biotechnology applications.

synthetic biology↗

bioRxiv2025

Advancing genetic engineering in the plant growth-promoting agricultural bacterium Azospirillum brasilense.

Azospirillum brasilense is an important plant-growth promoting bacterium found in the rhizosphere and used extensively in commercial agriculture. It is an attractive candidate for use in the development of novel crop interventions, such as the targeted delivery of plant hormones and similar compounds to the roots, shoots or leaves, or the introduction of smart bacterial sensors and actuators in the rhizosphere. In order to be able to engineer A. brasilense with these novel and complex behaviours, we require a collection of predictable and reliable genetic parts, consisting of constitutive and inducible promoters, terminators, and a stable expression plasmid. To date, such a genetic toolkit does not exist for the genus. In this work, for the first time we have designed and tested a synthetic constitutive promoter library with a wide-range of transcriptional strengths, a tightly-regulated inducible promoter with a non-metabolisable inducer, strong Rho-independent transcriptional terminators and synthetic small RNAs for post-transcriptional regulation.. We have adapted them for use in a one-pot assembly method and demonstrated their utility in the overproduction of the important plant hormone, indole 3-acetic acid (IAA).

synthetic biology↗

bioRxiv2024

Inhibition of glioblastoma cell proliferation and invasion by the choline-kinase inhibitor JAS239 varies with cell type and hypoxia

BackgroundElevated choline kinase alpha (ChoK) is observed in most solid tumours including glioblastomas (GBM), yet until recently, inhibitors of ChoK have demonstrated limited efficacy in GBM models. Given that hypoxia is associated with GBM therapy resistance, we hypothesised that tumour hypoxia could be responsible for such limitations. We therefore evaluated in GBM cells, the effect of hypoxia on the function of JAS239, a potent ChoK inhibitor. MethodsRodent (F98 and 9L) and human (U-87 MG and U-251 MG) GBM cell lines were subjected to 72 hours of hypoxia conditioning and treated with JAS239 for 24 hours. NMR metabolomic measurements and analyses were performed to evaluate the signalling pathways involved. In addition, cell proliferation, cell cycle progression and cell invasion were measured in cell monolayers and 3D spheroids, with or without JAS239 treatment in normoxic or hypoxic cells to assess how hypoxia affects JAS239 function. ResultsHypoxia and JAS239 treatment led to significant changes in the cellular metabolic pathways, specifically the phospholipid and glycolytic pathways associated with a reduction in cell proliferation via induced cell cycle arrest. Interestingly, JAS239 also impaired GBM invasion. However, JAS239 effects were variable depending on the cell line, reflecting the inherent heterogeneity observed in GBMs. ConclusionOur findings indicate that JAS239 and hypoxia can deregulate cellular metabolism, inhibit proliferation and alter cell invasion. These results may be useful for the design of new therapeutic strategies based on ChoK inhibition that can act on multiple pro-tumorigenic features.

cancer biology↗

bioRxiv2019

Transcriptional terminators allow leak-free chromosomal integration of genetic constructs in cyanobacteria

Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than model heterotrophic organisms, and the suite of well characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and insulate genes and operons from one another. Insulating gene expression can be particularly important when heterologous/synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium, Synechocystis sp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally-integrated rhamnose-inducible rhaBAD expression system from hypothesised read-through from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes.

synthetic biology↗

bioRxiv2017

A rhamnose-inducible system for precise and temporal control of gene expression in cyanobacteria

Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

synthetic biology↗

bioRxiv2017

Synthetic negative feedback circuits using engineered small RNAs

Negative feedback is known to endow biological and man-made systems with robust performance in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules which can inhibit translation of target messenger RNAs (mRNAs). In this paper, we designed, modelled and built two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA which negatively regulates the translation of the mRNA encoding this output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.

synthetic biology↗