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Baldwin, G. S.

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

2 recordsLinked to original sources

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

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