Biology subjects

Molloy, J. C.

Publications and source records attributed to Molloy, J. C..

6 recordsLinked to original sources

bioRxiv2025

Developing a Molecular Toolkit to ENABLE all to apply CRISPR/Cas9-based Gene Editing in planta

Gene editing, especially the CRISPR/Cas9 (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system, has revolutionized trait development in crops. However, large parts of the world are missing out on applying CRISPR in planta. There is an obvious lack of gene editing applications in locally relevant crops in the Global South which tend to be neglected by mainstream agricultural research and development. Access barriers to these new breeding technologies need to be removed to allow the potential impact of these technologies on food security to happen. Here, we present the ENABLE(R) Gene Editing in planta toolkit, a minimal molecular toolbox allowing users to create a CRISPR knockout vector for transient or stable plant transformation in two simple cloning steps. We validate the toolkit in rice (Oryza sativa) protoplasts and in Arabidopsis thaliana plants. The ENABLE(R) kit is designed to be utilized specifically by users in the Global South who are new to CRISPR technology by providing a simple workflow, extensive accompanying protocols as well as options for low cost methods for cloning verification and gene editing verification in planta. We hope that our toolkit helps bridge the gap between the recent biotechnological advancements in plant breeding that high income countries can access and the lack of those technologies in low and middle income countries.

plant biology↗

bioRxiv2024

qByte: Open-source isothermal fluorimeter for democratizing analysis of nucleic acids, proteins and cells

Access to affordable and reliable scientific instrumentation remains a significant barrier to the democratization of healthcare and scientific research. In the field of biotechnology, in particular, the complexity, cost, and infrastructure requirements of many instruments continue to limit their accessibility, especially in resource-limited environments. Despite the recent increase in the development of open-source tools, driven by advances in digital fabrication and electronic prototyping, few of these projects have reached large-scale implementation or validation in real-world settings. Here, we present qByte, an open-source, 8-tube isothermal fluorimeter designed to overcome these barriers by offering a cost-effective ($60) yet production-ready solution. qByte leverages standard digital manufacturing and Printed Circuit Board (PCB) assembly techniques and is designed to be portable, making it ideal for both laboratory and field use. The device has been benchmarked against commercial real-time thermocyclers and spectrophotometers, showing comparable results across four key applications: nucleic acid amplification and detection, analysis of protein activity and stability, genetic construct characterization, and bacterial viability tests. Further testing including the on-site diagnosis of human parasites in Kumasi, Ghana, and field work in Patagonia, Chile, validated qBytes reliability in real-world conditions. Taken together, our results proved qByte as flexible and reliable equipment for a variety of biological tests and applications, while its affordability and open-source design simplify further development and allow adaptation to the needs of future users.

bioengineering↗

bioRxiv2024

Semi-automated workflow for high-throughput Agrobacterium-mediated plant transformation

High-throughput experiments in plants are hindered by long generation times and high costs. To address these challenges, we present an optimized pipeline for Agrobacterium tumefaciens transformation and simplified a protocol to obtain stable transgenic lines of the model liverwort Marchantia polymorpha, paving the way for efficient high-throughput experiments for plant synthetic biology and other applications. Our protocol involves freeze-thaw Agrobacterium transformation method in 6-well plates that can be adapted to robotic automation. Using the Opentrons open-source platform, we implemented a semi-automated protocol showing similar efficiency compared to manual manipulation. Additionally, we have streamlined and simplified the process of stable transformation and selection of M. polymorpha, reducing cost, time, and manual labour without compromising transformation efficiency. The addition of sucrose in the selection media significantly enhances the production of gemmae, accelerating the generation of isogenic plants. We believe these protocols have the potential to facilitate high-throughput screenings in diverse plant species and represent a significant step towards the full automation of plant transformation pipelines. This approach allows testing [~]100 constructs per month, using conventional plant tissue culture facilities. We recently demonstrated the successful implementation of this protocol for screening hundreds of fluorescent reporters in Marchantia gemmae.

synthetic biology↗

bioRxiv2024

Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi

There is a critical need to implement a sensitive and specific point-of-care (POC) biosensor that addresses the instrument limitations and manufacturing challenges faced in resource-constrained contexts. In this paper we focus on enteric fever which is a highly contagious and prevalent infection in low- and middle-income countries. Although easily treatable, its ambiguous symptoms paired with a lack of fast, accurate and affordable diagnostics lead to incorrect treatments which exacerbate the disease burden, including increasing antibiotic resistance. In this study, we develop a readout module for CRISPR-Cas12a that produces a colorimetric output that is visible to the naked eye and can act as a cascade signal amplifier in any CRISPR assay based on trans-cleavage. We achieve this by immobilizing an oligo covalently linked to a {beta}-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. Upon cleavage, the colorimetric enzyme is released, and the supernatant transferred to an environment containing X-Gal producing an intense blue color. This method is capable of detecting amplified bacterial genomic DNA and has a lower limit of detection (LoD) to standard fluorescent assays while removing the requirement for costly equipment. Furthermore, it remained active after lyophilization, allowing for the possibility of shipment without cold chain, significantly reducing deployment costs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/606709v1_figSc1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d66787org.highwire.dtl.DTLVardef@2708eaorg.highwire.dtl.DTLVardef@ba5a86org.highwire.dtl.DTLVardef@8d4f48_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO Mechanism of CRISPR-Cas12a, and the signal output produced by enzyme trans-cleavage and the release of LacZ. 1) Initial set-up of inactivated system. 2) DNA isolation and amplification. 3) DNA addition to the biosensor. 4) CRISPR recognition of the DNA target and indiscriminate cleavage of all DNA. 5) LacZ release to the supernatant. 6) Transfer to an X-Gal environment. 7) X-Gal cleavage by LacZ and chromophore oxidation to the intensive blue indigo [illustration created with BioRender.com]. C_FIG

synthetic biology↗

bioRxiv2024

Open Educational Resources for distributed hands-on teaching in molecular biology

The urgent need to develop a more equitable bioeconomy has positioned biotechnology capacity building at the forefront of international priorities. However, in many educational institutions, particularly in low-and middle-income countries, this remains a major challenge due to limited access to reagents, equipment, and technical documentation. In this work, we describe Open Educational Resources (OER) composed of locally produced biological reagents, open source hardware and free software to teach fundamental techniques in biotechnology such as LAMP DNA amplification, RT-PCR RNA detection, enzyme kinetics and fluorescence imaging. The use of locally produced reagents and devices reduces costs by up to one order of magnitude. During the pandemic lockdowns, these tools were distributed nationwide to students homes as a lab-in-a-box for remote teaching of molecular biology. To test their performance, a total of 93 undergraduate students tested these resources during a biochemistry practical course. 27 out of 31 groups ([~]87%) successfully achieved the objectives of the PCR activity, while 28 out of 31 groups ([~]90%) correctly identified the target using LAMP reactions. To assess the potential application in secondary school, we organized three workshops for high school teachers from different institutions across Chile and performed an anonymous questionnaire, obtaining a strong agreement on how these OER broaden teachers perspectives on the techniques and facilitate the teaching of molecular biology topics. This effort was made possible through a close collaboration with open source technology advocates and members of DIYbio communities, whose work is paving the way for low-cost training in biology. All the protocols and design files are available under open source licenses.

molecular biology↗

bioRxiv2024

MobileLAMP: A portable, low-cost, open-source device for isothermal nucleic acid amplification

1Isothermal amplification-based methods for pathogen DNA or RNA detection offer high sensitivity, rapid detection, and the potential for deployment in remote fields and home testing. Consequently, they are emerging as alternatives to PCR and saw a surge in research activity and deployment for the rapid detection of SARS-CoV-2 during the Covid-19 pandemic. The most common isothermal DNA detection methods rely on minimal reagents for DNA amplification and simple hardware that can maintain isothermal conditions and read-out a fluorescent or colorimetric signal. Many researchers globally are working on improving these components based on diverse end-user needs. In this work, we have recognized the need for an open-source hardware device for isothermal amplification, composed of off-the-shelf components that are easily accessible in any part of the world, is easily manufacturable in a distributed and scalable way using 3D printing, and that can be powered using a wide diversity of batteries and power sources. We demonstrate the easy assembly of our device design and demonstrate its efficacy using colorimetric LAMP for both RNA and DNA targets.

bioengineering↗