Biology subjects

Garcia, D. C.

Publications and source records attributed to Garcia, D. C..

6 recordsLinked to original sources

bioRxiv2025

A neuron type-specific microexon in Ank3/ankyrin-G modulates calcium activity and neuronal excitability

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but included in cortical GABAergic neurons and cerebellar neurons, which is dictated by multiple neuronal splicing factors. In E35a-deletion mice we generated, interneurons show increased excitability and somatic Ca2+ activity, without disruption in AIS. Biochemical analyses suggest that E35a inclusion facilitates AnkG interaction with a protein complex involving inositol trisphosphate receptors (InsP3Rs) important for intracellular Ca2+ signaling. Alternative splicing therefore allows AnkG to modulate neuron type-specific excitability in addition to its ubiquitous pan-neuronal role in organizing the AIS.

neuroscience↗

bioRxiv2024

Semi-automated Production of Cell-free Biosensors

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including being able to be assembled, freeze-dried, distributed, and then used at the point-of-need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semi-automated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride sensing cell-free biosensors and demonstrate that they perform closely to expected detection outcomes.

synthetic biology↗

bioRxiv2024

High-Throughput Optimization of Paper-Based Cell-Free Biosensors

Cell-free expression systems maintain core cellular processes without intact cells and offer attractive properties as point-of-need biosensors. The ability to lyophilize, store, and use on-demand make these sensors usable in the field, and the lack of membranes means that there are no analyte transport issues and that new sensors can be deployed by simply adding a different DNA molecule. The lack of membranes also means that sensor designs and reaction optimizations can be screened in high throughput. While shelf stability has been demonstrated in specific cases using additives, these approaches are not universal to the myriad cell-free expression methods and formats. Here, we present new high-throughput screening methods to optimize cell-free expression formulations when embedded into paper for use as sensors. Our method leverages acoustic liquid handling to dispense reactions onto 384-well paper ticket formats and machine vision to quantify reaction performance from a colorimetric reporter enzyme. The throughput enabled shifts the bottleneck from experimental execution to selecting the experiments to execute; we therefore implement design-of-experiments to optimize the information gained from each design-build-test-learn cycle. We used these approaches to first optimize the performance of a low-cost cell-free expression formulation that was initially non-functional when embedded in paper, then further optimize it for tolerance to exposure to heat. With only 2 rounds of experimentation lasting 4 days total for each goal, the result are an energy mixture with 8% of the materials cost of a commonly used version and a formulation of excipients that maintain 60% of activity after 6 hours of storage at 50 {degrees}C and. Finally, we showcase the use of the cost-optimized formulation in a 3D-printed paperfluidic device where it outperforms the standard formulation at much lower cost.

synthetic biology↗

bioRxiv2023

Cell-Free Optimized Production of Protoporphyrin IX

BackgroundThe asymmetric and aromatic structures of porphyrins enable semiconducting properties allowing them to absorb light to initiate complex photocatalytic activity. These properties coupled with biological origins have garnered these molecules and their derivatives wide interest as a biotechnological platform. However, porphyrin production in cells is challenging due to the limited titer, long production timescales, and difficult purification. ResultsA cell-free metabolic engineering platform was constructed to produce protoporphyrin IX (PPIX) from E. coli crude extracts. Using acoustic liquid-handling, design of experiments for high-throughput buffer optimization and co-culturing techniques for extract production, our cell-free reactions effectively produced 0.109 mg/mL quantities of porphyrins. ConclusionsThe use of cell-free metabolic engineering as a bioproduction platform could improve the production of toxic or inefficient biomolecules such as PPIX. The engineering strategies including DOE and co-culturing applied in this study provide a roadmap towards increasing the scale of cell-free metabolic engineering by elucidating batch to batch variability, as well as the need for batch specific optimization of reaction conditions.

bioengineering↗

bioRxiv2021

A Method for Cost-Effective and Rapid Characterization of Genetic Parts

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a cost-effective and rapid method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house, as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR-amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components, or by titrating DNA concentrations rather than relying on limited plasmid copy numbers. Because this method retains a cell-like environment, the function of the genetic part will typically mimic its function in whole cells. SUMMARYWell-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

synthetic biology↗

bioRxiv2020

A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cells capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is limited. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective removal from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes are demonstrated that result in engineered crude lysates that are capable of 10 to 20-fold increases of pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to develop cell-free metabolic engineering into a practical platform for both bioprototyping and bioproduction. HighlightsO_LIA novel method of engineering crude cell lysates for enhancing specific metabolic processes is described. C_LIO_LIMultiplex Automated Genomic Engineering (MAGE) can be used to engineer donor strains for improving cell-free metabolite production with minimal impact on cell-growth. C_LIO_LIThe described lysate engineering strategy can specifically direct metabolic flux and create metabolic states not possible in living cells. C_LIO_LIPooling of the central precursor pyruvate was significantly improved through use of this lysate proteome engineering strategy. C_LI

synthetic biology↗