SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.

Soil microbiome from postmining ecosystems from Kabardino-Balkaria, Russia

<p>The soil microbiome is critical to the restoration of soils , destroyed by human activity. The dynamics of changes in the soil microbiome was investigated from the two overgrown gravel-sand quarry dumps in the North Caucasus (Kabardino-Balkaria, Russia). Samples were taken in the quarries of contrasting soil types (Calcareous Chernozem and Umbric Gleyic soils) under the various types of reclamation. Samples were taken from 10 points from a quarry with meadow soil and from 11 points from the Chernozem. The 16S ssu gene libraries were sequenced from soil DNA.The difference in microbiomes between the control points and the points where the soil is restored was statistically significant. The disturbed Gleyic soil is characterized by an increase in the representatives of Acidobacteria, for Chernozem of the genera <em>Niastella</em>, <em>Ramlibacter</em>, <em>Microvirga</em>. On the Umbric  Gleyic soil without reclamation, significant heterogeneity was shown, in contrast to Chernozem with different types of reclamation. In different soil types, the response of the soil microbiome to soil restoration was significantly different, which in turn should influence the choice of the strategy for the restoration of anthropogenically diturbed soils.</p>

DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions

Multiplexed assays allow functional testing of large synthetic libraries of genetic elements, but are limited by the designability, length, fidelity and scale of the input DNA. Here, we improve DropSynth, a low-cost, multiplexed method that builds gene libraries by compartmentalizing and assembling microarray-derived oligonucleotides in vortexed emulsions. By optimizing enzyme choice, adding enzymatic error correction and increasing scale, we show that DropSynth can build thousands of gene-length fragments at >20% fidelity.

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein. The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronaviruses has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naïve universal antibody gene libraries HAL9/10 anti-SARS-CoV-2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2 in the scFv-Fc format, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. All 17 scFv-Fc were able to bind the isolated RBD, four of them with sub-nanomolar EC50. Furthermore, these scFv-Fc neutralized active SARS-CoV-2 virus infection of VeroE6 cells. In a final step, the antibodies neutralizing best as scFv-Fc were converted into the IgG format. The antibody STE73-2E9 showed neutralization of active SARS-CoV-2 with an IC50 0.43 nM and is binding to the ACE2-RBD interface. Universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation.

A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries

Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene.

SINGLe: Accurate detection of single nucleotide polymorphisms using nanopore sequencing in gene libraries

Nanopore sequencing is a powerful single molecule DNA sequencing technology which offers high throughput and long sequence reads. Nevertheless, its high native error rate limits the direct detection of point mutations in individual reads of amplicon libraries, as these mutations are difficult to distinguish from the sequencing noise.In this work, we developed SINGLe (SNPs In Nanopore reads of Gene Libraries), a computational method to reduce the noise in nanopore reads of amplicons containing point variations. Our approach uses the fact that all reads are very similar to a wild type sequence, for which we experimentally characterize the position-specific systematic sequencing error pattern. We then use this information to reweight the confidence given to nucleotides that do not match the wild type in individual variant reads. We tested this method in a set of variants of KlenTaq, where the true mutation rate was well below the sequencing noise. SINGLe improves between 4 and 9 fold the signal to noise ratio, in comparison to the data returned by the basecaller guppy. Downstream, this approach improves variants clustering and consensus calling.SINGLe is simple to implement and requires only a few thousands reads of the wild type sequence of interest, which can be easily obtained by multiplexing in a single minION run. It does not require any modification in the experimental protocol, it does not imply a large loss of sequencing throughput, and it can be incorporated downstream of standard basecalling.

Coalescence of bacterial groups originating from urban runoffs and artificial infiltration systems among aquifer microbiomes

The invasion of aquifer microbial communities by aboveground micro-organisms, a phenomenon known as community coalescence, is likely to be exacerbated in groundwaters fed by stormwater infiltration systems (SIS). Here, the incidence of this increased connectivity with upslope soils and impermeabilized surfaces was assessed through a meta-analysis of 16S rRNA gene libraries. Specifically, free-living and attached aquifer bacteria (i.e., water and biofilm samples) were characterized upstream and downstream a SIS, and compared with bacterial communities from watershed runoffs, detention and infiltration basins. A significant bacterial transfer was observed, with aquifer bacterial biofilms being largely made up of taxa occurring in aboveground sediments and urban runoffs (44 to 67 % of the total reads). This coalesced biofilm community was rich in hydrocarbon degraders such as Sphingobium and Nocardia. The bacterial community of the downstream SIS aquifer waters showed similar coalescence with aboveground taxa (26.7–66.5 %) but a higher number of taxa involved in the N- and S-cycles was observed. A DNA marker named tpm enabled a tracking of bacterial species from 24 genera including the Pseudomonas, Aeromonas and Xanthomonas among these communities. Reads related to the Pseudomonas were allocated to 50 species, of which 16 were found in the aquifer samples. P. umsongensis and P. chengduensis were inferred to be in higher proportions among the tpm-harboring bacteria, respectively, of the aquifer biofilms, and waters. Several of these aquifer species were found involved in denitrification but also hydrocarbon degradation (P. aeruginosa, P. putida, and P. fluorescens). Reads related to Aeromonas were allocated to 11 species but only those from A. caviae were recovered in the aquifer samples. DNA imprints allocated to the X. axonopodis phytopathogen were recorded in higher proportions among the tpm-harboring bacteria of the aquifer waters than aboveground samples. A coalescence of microbial communities from an urban watershed with those of an aquifer was thus observed, and recent aquifer biofilms were found dominated by runoff opportunistic taxa able to use urban C-sources from aboveground compartments.

Antha-guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries

Protein engineering through directed evolution facilitates the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX™ liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries with minimal hands-on time and error-free, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads.

DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions

Multiplexed assays allow functional testing of large synthetic libraries of genetic elements, but are limited by the designability, length, fidelity and scale of the input DNA. Here we improve DropSynth, a low-cost, multiplexed method which builds gene libraries by compartmentalizing and assembling microarray-derived oligos in vortexed emulsions. By optimizing enzyme choice, adding enzymatic error correction, and increasing scale, we show that DropSynth can build thousands of gene-length fragments at >20% fidelity.