Biology subjects

Cremers, G.

Publications and source records attributed to Cremers, G..

4 recordsLinked to original sources

bioRxiv2023

Engineering a Scalable and Orthogonal Platform for Synthetic Communication in Mammalian Cells

The rational design and implementation of synthetic, orthogonal mammalian communication systems has the potential to unravel fundamental design principles of mammalian cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics and artificial tissue engineering. We lay here the foundations for the engineering of an orthogonal, and scalable mammalian synthetic intercellular communication platform that exploits the programmability of synthetic receptors and selective affinity and tunability of diffusing coiled-coil (CC) peptide heterodimers. Leveraging the ability of CCs to exclusively bind to a selected cognate receptor, we demonstrate orthogonal receptor activation, as well as Boolean logic computations. Next, we reveal synthetic intercellular communication based on synthetic receptors and secreted multidomain CC ligands and demonstrate a minimal, three-cell population system that can perform distributed AND gate logic. Our work provides a modular and scalable framework for the engineering of complex cell consortia, with the potential to expand the aptitude of cell therapeutics and diagnostics.

synthetic biology↗

bioRxiv2022

Microbial paracetamol degradation involves a high diversity of novel amidase enzyme candidates

Pharmaceuticals are relatively new to nature and often not completely removed in wastewater treatment plants (WWTPs). Consequently, these micropollutants end up in water bodies all around the world posing a great environmental risk. One exception to this recalcitrant conversion is paracetamol, whose full degradation has been linked to several microorganisms. However, the genes and corresponding proteins involved in microbial paracetamol degradation are still elusive. In order to improve our knowledge of the microbial paracetamol degradation pathway, we inoculated a bioreactor with sludge of a hospital WWTP (Pharmafilter, Delft, NL) and fed it with paracetamol as the sole carbon source. Paracetamol was fully degraded without any lag phase and the enriched microbial community was investigated by metagenomic and metatranscriptomic analyses, which demonstrated that the microbial community was very diverse. Dilution and plating on paracetamol-amended agar plates yielded two Pseudomonas sp. isolates: a fast-growing Pseudomonas sp. that degraded 200 mg/L of paracetamol in approximately 10 hours while excreting a dark brown component to the medium, and a slow-growing Pseudomonas sp. that degraded paracetamol without obvious intermediates in more than 90 days. Each Pseudomonas sp. contained a different highly-expressed amidase (31% identity to each other). These amidase genes were not detected in the bioreactor metagenome suggesting that other as-yet uncharacterized amidases may be responsible for the first biodegradation step of paracetamol. Uncharacterized deaminase genes and genes encoding dioxygenase enzymes involved in the catabolism of aromatic compounds and amino acids were the most likely candidates responsible for the degradation of paracetamol intermediates based on their high expression levels in the bioreactor metagenome and the Pseudomonas spp. genomes. Furthermore, cross-feeding between different community members might have occurred to efficiently degrade paracetamol and its intermediates in the bioreactor. This study increases our knowledge about the ongoing microbial evolution towards biodegradation of pharmaceuticals and points to a large diversity of (amidase) enzymes that are likely involved in paracetamol metabolism in WWTPs. HighlightsO_LIParacetamol was fully degraded by activated sludge from hospital wastewater. C_LIO_LILow paracetamol concentrations were removed by a diverse microbial community. C_LIO_LIPseudomonas sp. dominated cultures with high paracetamol concentration. C_LIO_LIUncharacterized amidases are probably involved in degrading paracetamol in WWTPs. C_LIO_LIDeaminases and dioxygenases might be degrading paracetamol transformation products. C_LI

microbiology↗

bioRxiv2020

Metagenomic profiling of ammonia- and methane-oxidizing microorganisms in a Dutch drinking water treatment plant

Elevated concentrations of ammonium and methane in groundwater can cause severe problems during drinking water production. To avoid their accumulation, raw water in the Netherlands, and many other countries, is purified by sand filtration. These drinking water filtration systems select for microbial communities that mediate the biodegradation of organic and inorganic compounds. In this study, the active layers and wall biofilm of a Dutch drinking water treatment plant (DWTP) were sampled at different locations along the filtration units of the plant over three years. We used high-throughput sequencing in combination with differential coverage and sequence composition-based binning to recover 56 near-complete metagenome-assembled genomes (MAGs) with an estimated completion of [≥]70% and with [≤]10% redundancy. These MAGs were used to characterize the microbial communities involved in the conversion of ammonia and methane. The methanotrophic microbial communities colonizing the wall biofilm (WB) and the granular material of the primary rapid sand filter (P-RSF) were dominated by members of the Methylococcaceae and Methylophilaceae. The abundance of these bacteria drastically decreased in the secondary rapid sand filter (S-RSF) samples. In all samples, complete ammonia-oxidizing (comammox) Nitrospira were the most abundant nitrifying guild. Clade A comammox Nitrospira dominated the P-RSF, while clade B was most abundant in WB and S-RSF, where ammonium concentrations were much lower. In conclusion, the knowledge obtained in this study contributes to understanding the role of microorganisms in the removal of carbon and nitrogen compounds during drinking water production. We furthermore found that drinking water treatment plants represent valuable model systems to study microbial community function and interaction. HighlightsO_LIMicrobial distribution was mainly influenced by sampling location within the DWTP C_LIO_LIClade A comammox Nitrospira were the dominant nitrifiers in the primary sand filter C_LIO_LIClade B was most abundant in samples from wall biofilm and the secondary filter C_LIO_LIA novel Methylophilaceae-affiliated methanotroph dominated the primary sand filter C_LI

microbiology↗

bioRxiv2019

Efficient small-scale conjugation of DNA to primary antibodies for multiplexed cellular targeting

The combination of the specificity of antibodies and the programmability of DNA nanotechnology has provided the scientific community with a powerful tool to label and unambiguously distinguish a large number of subcellular targets using fluorescence-based read-out methods. While primary antibodies are commercially available for a large class of targets, a general stoichiometric site-specific DNA labeling strategy for this affinity reagent is lacking. Here, we present a universal, site-selective, conjugation method using a small photocrosslinkable protein G adaptor that allows labeling of antibodies of different host species with a controlled number of short oligonucleotides (ODNs). Importantly, we illustrate that this conjugation method can be directly performed on commercially-available primary antibodies, on a small scale and without cross-reactivity towards other proteins, such as bovine serum albumin. In addition, we present a general, benchtop-compatible strategy to purify DNA-labeled antibodies without loss of function. The application of protein G-ODN labeled primary antibodies is demonstrated by employing three well-known methods for detecting subcellular targets using fluorescent read-out, including flow cytometry, DNA-PAINT, and dSTORM. This work thus establishes a general and efficient platform for the synthesis of a library of unique ODN-antibody conjugates, facilitating the broader use of DNA-based programmable tags for multiplexed labeling to identify subcellular features with nanometer-precision, improving our understanding of cellular structure and function.

biochemistry↗