Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multiplexed gene synthesis in emulsions for exploring protein functional landscapes.

ABSTRACT: Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene's assembly, which are then processed and assembled in water-in-oil emulsions. We used DropSynth to successfully build more than 7000 synthetic genes that encode phylogenetically diverse homologs of two essential genes in Escherichia coli We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout E. coli strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility. DropSynth coupled with multiplexed functional assays allows us to rationally explore sequence-function relationships at an unprecedented scale.

SUBMITTER: Plesa C

PROVIDER: S-EPMC6261299 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Multiplexed gene synthesis in emulsions for exploring protein functional landscapes.

Plesa Calin C Sidore Angus M AM Lubock Nathan B NB Zhang Di D Kosuri Sriram S

Science (New York, N.Y.) 20180104 6373

Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene's assembly, which are then processed and assembled in water-in-oil emulsions. We used DropSynth to successfully build more than 70 ...[more]

PMID: 29301959

Similar Datasets

DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions.

Project description: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.

| S-EPMC7498354 | biostudies-literature

Exploring protein fitness landscapes by directed evolution.

Project description:Directed evolution circumvents our profound ignorance of how a protein's sequence encodes its function by using iterative rounds of random mutation and artificial selection to discover new and useful proteins. Proteins can be tuned to adapt to new functions or environments by simple adaptive walks involving small numbers of mutations. Directed evolution studies have shown how rapidly some proteins can evolve under strong selection pressures and, because the entire 'fossil record' of evolutionary intermediates is available for detailed study, they have provided new insight into the relationship between sequence and function. Directed evolution has also shown how mutations that are functionally neutral can set the stage for further adaptation.

| S-EPMC2997618 | biostudies-literature

Exploring protein energy landscapes with hierarchical clustering.

Project description:In this work we present a new method for investigating local energy minima on a protein energy landscape. The CABS (CAlpha, CBeta and the center of mass of the Side chain) method was employed for generating protein models, but any other method could be used instead. Cα traces from an ensemble of models are hierarchical clustered with the HCPM (Hierarchical Clustering of Protein Models) method. The efficiency of this method for sampling and analyzing energy landscapes is shown.

| S-EPMC1366497 | biostudies-literature

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis.

Project description:Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

| S-EPMC9448781 | biostudies-literature

Exploring the energy landscapes of protein folding simulations with Bayesian computation.

Project description:Nested sampling is a Bayesian sampling technique developed to explore probability distributions localized in an exponentially small area of the parameter space. The algorithm provides both posterior samples and an estimate of the evidence (marginal likelihood) of the model. The nested sampling algorithm also provides an efficient way to calculate free energies and the expectation value of thermodynamic observables at any temperature, through a simple post processing of the output. Previous applications of the algorithm have yielded large efficiency gains over other sampling techniques, including parallel tempering. In this article, we describe a parallel implementation of the nested sampling algorithm and its application to the problem of protein folding in a G?-like force field of empirical potentials that were designed to stabilize secondary structure elements in room-temperature simulations. We demonstrate the method by conducting folding simulations on a number of small proteins that are commonly used for testing protein-folding procedures. A topological analysis of the posterior samples is performed to produce energy landscape charts, which give a high-level description of the potential energy surface for the protein folding simulations. These charts provide qualitative insights into both the folding process and the nature of the model and force field used.

| S-EPMC3283771 | biostudies-literature

A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes.

Project description:Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.

| S-EPMC4707994 | biostudies-literature

Developmental effector gene regulation: Multiplexed strategies for functional analysis.

Project description:The staggering complexity of the genome controls for developmental processes is revealed through massively parallel cis-regulatory analysis using new methods of perturbation and readout. The choice of combinations of these new methods is tailored to the system, question and resources at hand. Our focus is on issues that include the necessity or sufficiency of given cis-regulatory modules, cis-regulatory function in the normal spatial genomic context, and easily accessible high throughput and multiplexed analysis methods. In the sea urchin embryonic model, recombineered BACs offer new opportunities for consecutive modes of cis-regulatory analyses that answer these requirements, as we here demonstrate on a diverse suite of previously unstudied sea urchin effector genes expressed in skeletogenic cells. Positively active cis-regulatory modules were located in single Nanostring experiments per BAC containing the gene of interest, by application of our previously reported "barcode" tag vectors of which> 100 can be analyzed at one time. Computational analysis of DNA sequences that drive expression, based on the known skeletogenic regulatory state, then permitted effective identification of functional target site clusters. Deletion of these sub-regions from the parent BACs revealed module necessity, as simultaneous tests of the same regions in short constructs revealed sufficiency. Predicted functional inputs were then confirmed by site mutations, all generated and tested in multiplex formats. There emerged the simple conclusion that each effector gene utilizes a small subset of inputs from the skeletogenic GRN. These inputs may function to only adjust expression levels or in some cases necessary for expression. Since we know the GRN architecture upstream of the effector genes, we could then conceptually isolate and compare the wiring of the effector gene driver sub-circuits and identify the inputs whose removal abolish expression.

| S-EPMC6321769 | biostudies-literature

Exploring functional roles of multibinding protein interfaces.

Project description:Cellular processes are highly interconnected and many proteins are shared in different pathways. Some of these shared proteins or protein families may interact with diverse partners using the same interface regions; such multibinding proteins are the subject of our study. The main goal of our study is to attempt to decipher the mechanisms of specific molecular recognition of multiple diverse partners by promiscuous protein regions. To address this, we attempt to analyze the physicochemical properties of multibinding interfaces and highlight the major mechanisms of functional switches realized through multibinding. We find that only 5% of protein families in the structure database have multibinding interfaces, and multibinding interfaces do not show any higher sequence conservation compared with the background interface sites. We highlight several important functional mechanisms utilized by multibinding families. (a) Overlap between different functional pathways can be prevented by the switches involving nearby residues of the same interfacial region. (b) Interfaces can be reused in pathways where the substrate should be passed from one protein to another sequentially. (c) The same protein family can develop different specificities toward different binding partners reusing the same interface; and finally, (d) inhibitors can attach to substrate binding sites as substrate mimicry and thereby prevent substrate binding.

| S-EPMC2776955 | biostudies-literature

Iterative, multiplexed CRISPR-mediated gene editing for functional analysis of complex protease gene clusters.

Project description:Elucidation of gene function by reverse genetics in animal models frequently is complicated by the functional redundancy of homologous genes. This obstacle often is compounded by the tight clustering of homologous genes, which precludes the generation of multigene-deficient animals through standard interbreeding of single-deficient animals. Here, we describe an iterative, multiplexed CRISPR-based approach for simultaneous gene editing in the complex seven-member human airway trypsin-like protease/differentially expressed in a squamous cell carcinoma (HAT/DESC) cluster of membrane-anchored serine proteases. Through four cycles of targeting, we generated a library of 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Using this library, we demonstrate that HAT/DESC proteases are dispensable for term development, postnatal health, and fertility and that the recently described function of the HAT-like 4 protease in epidermal barrier formation is unique among all HAT/DESC proteases. The study demonstrates the potential of iterative, multiplexed CRISPR-mediated gene editing for functional analysis of multigene clusters, and it provides a large array of new congenic mouse strains for the study of HAT/DESC proteases in physiological and in pathophysiological processes.

| S-EPMC6827298 | biostudies-literature

Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen.

Project description:The use of small molecule inhibitors of cellular processes is a powerful approach to understanding gene function that complements the genetic approach. We have designed a high throughput screen to identify new inhibitors of eukaryotic protein synthesis. We used a bicistronic mRNA reporter to multiplex our assay and simultaneously screen for inhibitors of cap-dependent initiation, internal initiation and translation elongation/termination. Functional screening of >90 000 compounds in an in vitro translation reaction identified 36 inhibitors, 14 of which are known inhibitors of translation and 18 of which are nucleic acid-binding ligands. Our results indicate that intercalators constitute a large class of protein synthesis inhibitors. Four non-intercalating compounds were identified, three of which block elongation and one of which inhibits initiation. The novel inhibitor of initiation affects 5' end-mediated initiation, as well as translation initiated from picornaviral IRESs, but does not significantly affect internal initiation from the hepatitis C virus 5'-untranslated region. This compound should be useful for delineating differences in mechanism of initiation among IRESs.

| S-EPMC373382 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data