An open-source automated PEG precipitation assay to measure the relative solubility of proteins with low material requirement

The solubility of proteins correlates with a variety of their properties, including function, production yield, pharmacokinetics, and formulation at high concentrations. High solubility is therefore a key requirement for the development of protein-based reagents for applications in life sciences, biotechnology, diagnostics, and therapeutics. Accurate solubility measurements, however, remain challenging and resource intensive, which limits their throughput and hence their applicability at the early stages of development pipelines, when long-lists of candidates are typically available in minute amounts. Here, we present an automated method based on the titration of a crowding agent (polyethylene glycol, PEG) to quantitatively assess relative solubility of proteins using about 200 µg of purified material. Our results demonstrate that this method is accurate and economical in material requirement and costs of reagents, which makes it suitable for high-throughput screening. This approach is freely-shared and based on a low cost, open-source liquid-handling robot. We anticipate that this method will facilitate the assessment of the developability of proteins and make it substantially more accessible.

Semi-automated high-throughput substrate screening assay for nucleoside kinases

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and the production of nucleotide analogues in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening assay for NKs is of great importance. Here, we report the validation of a well-known luciferase-based assay for the detection of NK activity in 96-well plate format. The assay was semi-automated using a liquid handling robot. A good linearity was demonstrated (r² >0.98) in the range of 0 to 500 µM ATP, and it was shown that also alternative phosphate donors like dATP or CTP were accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplary used for the comparison of the substrate spectra of four nucleoside kinases using 20 (8 natural and 12 modified) substrates. The screening results correlated well with literature data and, additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

Implementation of an efficient SARS-CoV-2 specimen pooling strategy for high throughput diagnostic testing

The rapid identification and isolation of infected individuals remains a key strategy for controlling the spread of SARS-CoV-2. Frequent testing of populations to detect infection early in asymptomatic or presymptomatic individuals can be a powerful tool for intercepting transmission, especially when the viral prevalence is low. However, RT-PCR testing—the gold standard of SARS-CoV-2 diagnosis—is expensive, making regular testing of every individual unfeasible. Sample pooling is one approach to lowering costs. By combining samples and testing them in groups the number of tests required is reduced, substantially lowering costs. Here we report on the implementation of pooling strategies using 3-d and 4-d hypercubes to test a professional sports team in South Africa. We have shown that infected samples can be reliably detected in groups of 27 and 81, with minimal loss of assay sensitivity for samples with individual Ct values of up to 32. We report on the automation of sample pooling, using a liquid-handling robot and an automated web interface to identify positive samples. We conclude that hypercube pooling allows for the reliable RT-PCR detection of SARS-CoV-2 infection, at significantly lower costs than lateral flow antigen (LFA) tests.

Bead preparation protocol v1

STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. Here we describe the protocol to prepare the magnetic beads that are currently used in STRIP. Note, in this protocol we use Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic) from Cytivia, but other beads such as SeraSil Mag 400 beads (Cytiva) can be used as substitutes.

Step 2: RNA extraction and RT-qPCR v1

STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. Key features of the customized Tecan Fluent robot are (1) on-board 1D and 2D barcode scanners, (2) an automated tube decapper, (3) two robotic arms for simultaneous processing of different procedural steps, (4) a newly-designed spin vessel to keep magnetic beads in solution and immediately transferable to 384-well plates, (5) a built-in magnetic deck and a 384-channel pipetting head for rapid RNA isolation, (6) a heating device for fast drying of RNA prior to elution, (7) a built-in plate sealer and (8) a plate storage system to allow processing of multiple sample plates in a single run (See Materials). Here we describe the RNA extraction and RT-qPCR protocol as currently applied in STRIP.

Open-source personal pipetting robots with live-cell incubation and microscopy compatibility

Liquid handling robots have the potential to automate many procedures in life sciences. However, they are not in widespread use in academic settings, where funding, space and maintenance specialists are usually limiting. In addition, current robots require lengthy programming by specialists and are incompatible with most academic laboratories with constantly changing small-scale projects. Here, we present the Pipetting Helper Imaging Lid (PHIL), an inexpensive, small, open-source personal liquid handling robot. It is designed for inexperienced users, with self-production from cheap commercial and 3D-printable components and custom control software. PHIL successfully automated pipetting for e.g. tissue immunostainings and stimulations of live stem and progenitor cells during time-lapse microscopy. PHIL is cheap enough for any laboratory member to have their own personal pipetting robot(s), and enables users without programming skills to easily automate a large range of experiments.

An open-source semi-automated robotics pipeline for embryo immunohistochemistry

A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts. Large-scale developmental screens such as unbiased mutagenesis surveys have been limited in their applicability to embryonic systems, as the technologies for quantifying precise expression patterns in whole animals has not kept pace with other sequencing-based technologies. Here, we outline an open-source semi-automated pipeline to chemically fixate, stain, and 3D-image Drosophila embryos. Central to this pipeline is a liquid handling robot, Flyspresso, which automates the steps of classical embryo fixation and staining. We provide the schematics and an overview of the technology for an engineer or someone equivalently trained to reproduce and further improve upon Flyspresso, and highlight the Drosophila embryo fixation and colorimetric or antibody staining protocols. Additionally, we provide a detailed overview and stepwise protocol for our adaptive-feedback pipeline for automated embryo imaging on confocal microscopes. We demonstrate the efficiency of this pipeline compared to classical techniques, and how it can be repurposed or scaled to other protocols and biological systems. We hope our pipeline will serve as a platform for future research, allowing a broader community of users to build, execute, and share similar experiments.

Implementation of an efficient SARS-CoV-2 specimen pooling strategy for high throughput diagnostic testing

The rapid identification and isolation of infected individuals remains a key strategy for controlling the spread of SARS-CoV-2. Frequent testing of populations to detect infection early in asymptomatic or presymptomatic individuals can be a powerful tool for intercepting transmission, especially when the viral prevalence is low. However, RT-PCR testing – the gold standard of SARS-CoV-2 diagnosis – is expensive, making regular testing of every individual unfeasible. Sample pooling is one approach to lowering costs. By combining samples and testing them in groups the number of tests required is reduced, substantially lowering costs. Here we report on the implementation of pooling strategies using 3-d and 4-d hypercubes to test a professional sports team in South Africa. We have shown that infected samples can be reliably detected in groups of 27 and 81, with minimal loss of assay sensitivity for samples with individual Ct values up to 32. We report on automation of sample pooling, using a liquid-handling robot and an automated web interface to identify positive samples. We conclude that hypercube pooling allows for the reliable RT-PCR detection of SARS-CoV-2 infection, at significantly lower cost than lateral flow antigen (LFA) tests.

Predicting population genetic change in an experimental stochastic environment

Most natural environments exhibit a substantial component of random variation. Such environmental noise is expected to cause random fluctuations in natural selection, affecting the predictability of evolution. But despite a long-standing theoretical interest for understanding the population genetic consequences of stochastic environments, there has been a dearth of empirical validation and estimation of the underlying parameters of this theory. Indeed, tracking the genetics of a large number of replicate lines under a controlled level of environmental stochasticity is particularly challenging. Here, we tackled this problem by resorting to an automated experimental evolution approach. We used a liquid-handling robot to expose over a hundred lines of the micro-alga Dunaliella salina to randomly fluctuating salinity over a continuous range, with controlled mean, variance, and autocorrelation. We then tracked the frequency of one of two competing strains through amplicon sequencing of a nuclear and choloroplastic barcode sequences. We show that the magnitude of environmental fluctuations (variance), but also their predictability (autocorrelation), have large impacts on the average selection coefficient. Furthermore, the stochastic variance in population genetic change is substantially higher in a fluctuating environment. Reaction norms of selection coefficients and growth rates of single strains against the environment captured the mean response accurately, but failed to explain the high variance induced by environmental stochasticity. Overall, our results provide exceptional insights on the prospects for understanding and predicting genetic evolution in randomly fluctuating environments.

Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection

Background Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated. Methods One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control. Results No significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350€ and 400€ for every 96 extracted samples compared to the commercial kit. Conclusion The protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities.