Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format.

Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format.

Mass spectrometry imaging-based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme.

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data.

A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria.

Exometabolomics is an approach to assess how microorganisms alter, or react to their environments through the depletion and production of metabolites. It allows the examination of how soil microbes transform the small molecule metabolites within their environment, which can be used to study resource competition and cross-feeding. This approach is most powerful when used with defined media that enable tracking of all metabolites.

Biofilm Interaction Mapping and Analysis (BIMA) of Interspecific Interactions in Co-culture Biofilms.

Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of biofilms, in combination with consortia-based approaches, may offer advantages for these processes.

Faster, better, and cheaper: harnessing microfluidics and mass spectrometry for biotechnology.

High-throughput screening technologies are widely used for elucidating biological activities. These typically require trade-offs in assay specificity and sensitivity to achieve higher throughput. Microfluidic approaches enable rapid manipulation of small volumes and have found a wide range of applications in biotechnology providing improved control of reaction conditions, faster assays, and reduced reagent consumption.

Cervicovaginal Microbiome Composition Is Associated with Metabolic Profiles in Healthy Pregnancy.

Microbes and their metabolic products influence early-life immune and microbiome development, yet remain understudied during pregnancy. Vaginal microbial communities are typically dominated by one or a few well-adapted microbes which are able to survive in a narrow pH range and are adapted to live on host-derived carbon sources, likely sourced from glycogen and mucin present in the vaginal environment. We characterized the cervicovaginal microbiomes of 16 healthy women throughout the three trimesters of pregnancy.

Root morphology and exudate availability are shaped by particle size and chemistry in .

Root morphology and exudation define a plants' sphere of influence in soils. In turn, soil characteristics influence plant growth, morphology, root microbiome, and rhizosphere chemistry. Collectively, all these parameters have significant implications on the major biogeochemical cycles, crop yield, and ecosystem health.

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria.

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts.

A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts.

Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high-throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high-throughput mass-spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step.

Lessons from Two Design-Build-Test-Learn Cycles of Dodecanol Production in Escherichia coli Aided by Machine Learning.

The Design-Build-Test-Learn (DBTL) cycle, facilitated by exponentially improving capabilities in synthetic biology, is an increasingly adopted metabolic engineering framework that represents a more systematic and efficient approach to strain development than historical efforts in biofuels and biobased products. Here, we report on implementation of two DBTL cycles to optimize 1-dodecanol production from glucose using 60 engineered Escherichia coli MG1655 strains. The first DBTL cycle employed a simple strategy to learn efficiently from a relatively small number of strains (36), wherein only the choice of ribosome-binding sites and an acyl-ACP/acyl-CoA reductase were modulated in a single pathway operon including genes encoding a thioesterase (UcFatB1), an acyl-ACP/acyl-CoA reductase (Maqu_2507, Maqu_2220, or Acr1), and an acyl-CoA synthetase (FadD).

Enzyme promiscuity shapes adaptation to novel growth substrates.

Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations.

MAGI: A Method for Metabolite Annotation and Gene Integration.

Metabolomics is a widely used technology for obtaining direct measures of metabolic activities from diverse biological systems. However, ambiguous metabolite identifications are a common challenge and biochemical interpretation is often limited by incomplete and inaccurate genome-based predictions of enzyme activities (that is, gene annotations). Metabolite Annotation and Gene Integration (MAGI) generates a metabolite-gene association score using a biochemical reaction network.

Insulator Nanostructure Desorption Ionization Mass Spectrometry.

Surface-assisted laser desorption ionization (SALDI) is an approach for gas-phase ion generation for mass spectrometry using laser excitation on typically conductive or semiconductive nanostructures. Here, we introduce insulator nanostructure desorption ionization mass spectrometry (INDI-MS), a nanostructured polymer substrate for SALDI-MS analysis of small molecules and peptides. INDI-MS surfaces are produced through the self-assembly of a perfluoroalkyl silsesquioxane nanostructures in a single chemical vapor deposition silanization-step.

Morphology-Driven Control of Metabolite Selectivity Using Nanostructure-Initiator Mass Spectrometry.

Nanostructure-initiator mass spectrometry (NIMS) is a laser desorption/ionization analysis technique based on the vaporization of a nanostructure-trapped liquid "initiator" phase. Here we report an intriguing relationship between NIMS surface morphology and analyte selectivity. Scanning electron microscopy and spectroscopic ellipsometry were used to characterize the surface morphologies of a series of NIMS substrates generated by anodic electrochemical etching.

OpenMSI Arrayed Analysis Toolkit: Analyzing Spatially Defined Samples Using Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) has primarily been applied in localizing biomolecules within biological matrices. Although well-suited, the application of MSI for comparing thousands of spatially defined spotted samples has been limited. One reason for this is a lack of suitable and accessible data processing tools for the analysis of large arrayed MSI sample sets.

Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay.

Currently, the identification of new genes drastically outpaces current experimental methods for determining their enzymatic function. This disparity necessitates the development of high-throughput techniques that operate with the same scalability as modern gene synthesis and sequencing technologies. In this paper, we demonstrate the versatility of the recently reported DNA-Linked Enzyme-Coupled Assay (DLEnCA) and its ability to support high-throughput data acquisition through next-generation sequencing (NGS).

Application of Black Silicon for Nanostructure-Initiator Mass Spectrometry.

Nanostructure-initiator mass spectrometry (NIMS) is a matrix-free desorption/ionization technique with high sensitivity for small molecules. Surface preparation has relied on hydrofluoric acid (HF) electrochemical etching which is undesirable given the significant safety controls required in this specialized process. In this study, we examine a conventional and widely used process for producing black silicon based on sulfur hexafluoride/oxygen (SF6/O2) inductively coupled plasma (ICP) etching at cryogenic temperatures and we find it to be suitable for NIMS.

High-throughput platforms for metabolomics.

Mass spectrometry has become a choice method for broad-spectrum metabolite analysis in both fundamental and applied research. This can range from comprehensive analysis achieved through time-consuming chromatography to the rapid analysis of a few target metabolites without chromatography. In this review article, we highlight current high-throughput MS-based platforms and their potential application in metabolomics.

DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity.

Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification.

Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers.

The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers.

Production and biomedical applications of virus-like particles derived from polyomaviruses.

Virus-like particles (VLPs), aggregates of capsid proteins devoid of viral genetic material, show great promise in the fields of vaccine development and gene therapy. These particles spontaneously self-assemble after heterologous expression of viral structural proteins. This review will focus on the use of virus-like particles derived from polyomavirus capsid proteins.

A solid-phase platform for combinatorial and scarless multipart gene assembly.

With the emergence of standardized genetic modules as part of the synthetic biology toolbox, the need for universal and automatable assembly protocols increases. Although several methods and standards have been developed, these all suffer from drawbacks such as the introduction of scar sequences during ligation or the need for specific flanking sequences or a priori knowledge of the final sequence to be obtained. We have developed a method for scarless ligation of multipart gene segments in a truly sequence-independent fashion.