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Biology subjects

Howard, T. P.

Publications and source records attributed to Howard, T. P..

4 recordsLinked to original sources

OT-Mation: an open-source code for parsing CSV files into python scripts for control of OT-2 liquid handling robotics

OT-Mation is an open-source Python script designed to automate the programming of OT-2 liquid handling robots, making combinatorial experiments more accessible to researchers. By parsing user-defined CSV files containing information on labware, reagents, pipettes, and experimental design, OT-Mation generates a bespoke Python script compatible with the OT-2 system. OT-Mation enhances reproducibility, reduces human error, and streamlines workflows, making it a valuable addition to any laboratory utilising OT-2 robotics for liquid handling. While OT-Mation can be used for setting up any type of experiment on the OT-2, its real utility lies in making the connection between multifactorial experimental design software outputs (i.e. Design of Experiments arrays) and liquid-handling robot executable code. As such, OT-Mation helps bridge the gap between code-based flexibility and user-friendly operation, allowing researchers with limited programming skills to design and execute complex experiments efficiently.

synthetic biology↗

Cell-free genetic devices confer autonomic and adaptive properties to hydrogels

Smart materials are able to alter one or more of their properties in response to defined stimuli. Our ability to design and create such materials, however, does not match the diversity and specificity of responses seen within the biological domain. We propose that relocation of molecular phenomena from living cells into hydrogels can be used to confer smart functionality to materials. We establish that cell-free protein synthesis can be conducted in agarose hydrogels, that gene expression occurs throughout the material and that co-expression of genes is possible. We demonstrate that gene expression can be controlled transcriptionally (using in gel gene interactions) and translationally in response to small molecule and nucleic acid triggers. We use this system to design and build a genetic device that can alter the structural property of its chassis material in response to exogenous stimuli. Importantly, we establish that a wide range of hydrogels are appropriate chassis for cell-free synthetic biology, meaning a designer may alter both the genetic and hydrogel components according to the requirements of a given application. We probe the relationship between the physical structure of the gel and in gel protein synthesis and reveal that the material itself may act as a macromolecular crowder enhancing protein synthesis. Given the extensive range of genetically encoded information processing networks in the living kingdom and the structural and chemical diversity of hydrogels, this work establishes a model by which cell-free synthetic biology can be used to create autonomic and adaptive materials. Significance statementSmart materials have the ability to change one or more of their properties (e.g. structure, shape or function) in response to specific triggers. They have applications ranging from light-sensitive sunglasses and drug delivery systems to shape-memory alloys and self-healing coatings. The ability to programme such materials, however, is basic compared to the ability of a living organism to observe, understand and respond to its environment. Here we demonstrate the relocation of biological information processing systems from cells to materials. We achieved this by operating small, programmable genetic devices outside the confines of a living cell and inside hydrogel matrices. These results establish a method for developing materials functionally enhanced with molecular machinery from biological systems.

synthetic biology↗

Data Driven Modelling of a Chemically Defined Growth Medium for Cupriavidus necator H16

Cupriavidus necator is a Gram-negative soil bacterium of major biotechnological interest. It is a producer of the bioplastic 3-polyhydroxybutyrate, has been exploited in bioremediation processes, and its lithoautotrophic capabilities suggest it may function as a microbial cell factory upgrading renewable resources to fuels and chemicals. It remains necessary however to develop appropriate experimental resources to permit controlled bioengineering and system optimisation of this microbial chassis. A key resource for physiological, biochemical and metabolic studies of any microorganism is a chemically defined growth medium. Here we use 1 mL micro-well cell cultures, automated liquid handling and a statistical engineering approach to develop a model that describes the effect of key media components and their interactions on C. necator culture cell density. The model is predictive and was experimentally validated against novel media compositions. Moreover, the model was further validated against larger culture volumes at 100 mL and 1 L volumes and found to correlate well. This approach provides valuable and quantifiable insights into the impact of media components on cell growth as well as providing predictions to guide culture scale-up.

microbiology↗

Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition

Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. Through direct-to-patient outreach, we developed genomically faithful patient-derived models of RMC. Using whole genome sequencing, we identified intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele, confirming that SMARCB1 loss occurs in RMC. Biochemical and functional characterization of these RMC models revealed that RMC depends on the loss of SMARCB1 for survival and functionally resemble other cancers that harbor loss of SMARCB1, such as malignant rhabdoid tumors or atypical teratoid rhabdoid tumors. We performed RNAi and CRISPR-Cas9 loss of function genetic screens and a small-molecule screen and identified UBE2C as an essential gene in SMARCB1 deficient cancers. We found that the ubiquitin-proteasome pathway was essential for the survival of SMARCB1 deficient cancers in vitro and in vivo. Genetic or pharmacologic inhibition of this pathway leads to G2/M arrest due to constitutive accumulation of cyclin B1. These observations identify a synthetic lethal relationship that may serve as a therapeutic approach for patients with SMARCB1 deficient cancers.

cancer biology↗