Biology subjects

Baldwin, G. S.

Publications and source records attributed to Baldwin, G. S..

6 recordsLinked to original sources

bioRxiv2024

Fine Tuning Genetic Circuits via Host Context and RBS Modulation

The choice of organism to host a genetic circuit - the chassis - is often defaulted to model organisms due to their amenability. The chassis-design space has therefore remained underexplored as an engineering variable. In this work, we explored the design space of a genetic toggle switch through variations in nine ribosome binding sites compositions and three host contexts, creating 27 circuit variants. Characterization of performance metrics in terms of toggle switch output and host growth dynamics unveils a spectrum of performance profiles from our circuit library. We find that changes in host-context causes large shifts in overall performance, while modulating ribosome binding sites leads to more incremental changes. We find that a combined ribosome binding site and host-context modulation approach can be used to fine tune the properties of a toggle switch according to user-defined specifications, such as towards greater signaling strength, inducer sensitivity or both. Other auxiliary properties, such as inducer tolerance, are also exclusively accessed through changes in host-context. We demonstrate here that exploration of the chassis-design space can offer significant value, reconceptualizing the chassis-organism as an important part in the synthetic biologists toolbox with important implications for the field of synthetic biology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/604438v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1ff45d0org.highwire.dtl.DTLVardef@4060d7org.highwire.dtl.DTLVardef@95d5d8org.highwire.dtl.DTLVardef@10a98d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Revealing the chassis-effect on a broad-host-range genetic switch and its concordance with interspecies bacterial physiologies

Broad-host-range synthetic biology is an emerging frontier that aims to expand our current engineerable domain of microbial hosts for biodesign applications. As more novel species are brought to "model status", synthetic biologists are discovering that identically engineered genetic circuits can exhibit different performances depending on the organism it operates within, an observation referred to as the "chassis-effect". It remains a major challenge to uncover which genome encoded and physiological biological determinants will underpin chassis effects that govern the performance of engineered genetic devices. In this study, we compared model and novel bacterial hosts to ask whether phylogenomic relatedness or similarity in host physiology is a better predictor of toggle switch performance. This was accomplished using comparative framework based on multivariate statistical approaches to systematically demonstrate the chassis-effect and characterize the performance dynamics of a genetic toggle switch operating within six Gammaproteobacteria. Our results solidify the notion that genetic devices are significantly impacted by host-context. Furthermore, we formally determined that hosts exhibiting more similar metrics of growth and molecular physiology also exhibit more similar toggle switch performance, indicating that specific bacterial physiology underpins measurable chassis effects. The result of this study contributes to the field of broad-host-range synthetic biology by lending increased predictive power to the implementation of genetic devices in less-established microbial hosts.

synthetic biology↗

bioRxiv2021

A modular toolset for electrogenetics

Synthetic biology research and its industrial applications rely on the deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and an electrode), allowing for the direct integration of electronics with complex biological processes for a variety of new applications. However, the use of electrogenetic systems is limited by poor activity, tunability and standardisation. Here, we have developed a variety of genetic and electrochemical tools that facilitate the design and vastly improve the performance of electrogenetic systems. We developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We then constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Finally, we demonstrated electrochemical activation of gene expression in aerobic conditions utilising a novel, modular bioelectrochemical device. This toolset provides researchers with all the elements needed to design and build optimised electrogenetic systems for specific applications.

synthetic biology↗

bioRxiv2021

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.

microbiology↗

bioRxiv2019

DNA-BOT: A low-cost, automated DNA assembly platform for synthetic biology

Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems that perform the desired functions. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process has received serious attention in recent years. Importantly, automating DNA assembly enables larger builds using less researcher time, increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package dnabot (https://github.com/BASIC-DNA-ASSEMBLY/dnabot). We demonstrate the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, exploring the promoter, ribosome binding site (RBS) and gene order design space for a 3-gene operon. All 88 constructs were assembled with high accuracy, at a cost of $1.50 - $5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT making it accessible for most labs and democratising automated DNA assembly.

synthetic biology↗

bioRxiv2019

Robust Estimation of Bacterial Cell Count from Optical Density

Optical density (OD) is a fast, cheap, and high-throughput measurement widely used to estimate the density of cells in liquid culture. These measurements, however, cannot be compared between instruments without a standardized calibration protocol and are challenging to relate to actual cell count. We address these shortcomings with an interlaboratory study comparing three OD calibration protocols, as applied to eight strains of E. coli engineered to constitutively express varying levels of GFP. These three protocols--comparison with colloidal silica (LUDOX), serial dilution of silica microspheres, and a reference colony-forming unit (CFU) assay--are all simple, low-cost, and highly accessible. Based on the results produced by the 244 teams completing this interlaboratory study, we recommend calibrating OD using serial dilution of silica microspheres, which readily produces highly precise calibration (95.5% of teams having residuals less than 1.2-fold), is easily assessed for quality control, and as a side effect also assesses the effective linear range of an instrument. Moreover, estimates of cell count from silica microspheres can be combined with fluorescence calibration against fluorescein to obtain units of Molecules of Equivalent Fluorescein (MEFL), allowing direct comparison and data fusion with equivalently calibrated flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.

synthetic biology↗