scholarly journals Rapid and scalable preparation of bacterial lysates for cell-free gene expression

2017 ◽  
Author(s):  
Andriy Didovyk ◽  
Taishi Tonooka ◽  
Lev Tsimring ◽  
Jeff Hasty
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Cost Effective ◽  
Diverse Range ◽  
Highly Active ◽  
Freeze Dried ◽  
Wide Range ◽  
Specialized Equipment ◽  
Free Gene ◽  
Bacterial Lysates

AbstractCell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, ro-bust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. More-over, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by reg-ulating protein production and degradation, implementing quorum sensing, and showing quan-titative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

scholarly journals Cell-free genetic devices confer autonomic and adaptive properties to hydrogels

2019 ◽  
Author(s):  
Colette J. Whitfield ◽  
Alice M. Banks ◽  
Gema Dura ◽  
John Love ◽  
Jonathan E. Fieldsend ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Information Processing ◽  
Synthetic Biology ◽  
Smart Materials ◽  
Living Organism ◽  
Chemical Diversity ◽  
Biological Information ◽  
Self Healing ◽  
Wide Range

AbstractSmart materials are able to alter one or more of their properties in response to defined stimuli. Our ability to design and create such materials, however, does not match the diversity and specificity of responses seen within the biological domain. We propose that relocation of molecular phenomena from living cells into hydrogels can be used to confer smart functionality to materials. We establish that cell-free protein synthesis can be conducted in agarose hydrogels, that gene expression occurs throughout the material and that co-expression of genes is possible. We demonstrate that gene expression can be controlled transcriptionally (using in gel gene interactions) and translationally in response to small molecule and nucleic acid triggers. We use this system to design and build a genetic device that can alter the structural property of its chassis material in response to exogenous stimuli. Importantly, we establish that a wide range of hydrogels are appropriate chassis for cell-free synthetic biology, meaning a designer may alter both the genetic and hydrogel components according to the requirements of a given application. We probe the relationship between the physical structure of the gel and in gel protein synthesis and reveal that the material itself may act as a macromolecular crowder enhancing protein synthesis. Given the extensive range of genetically encoded information processing networks in the living kingdom and the structural and chemical diversity of hydrogels, this work establishes a model by which cell-free synthetic biology can be used to create autonomic and adaptive materials.Significance statementSmart materials have the ability to change one or more of their properties (e.g. structure, shape or function) in response to specific triggers. They have applications ranging from light-sensitive sunglasses and drug delivery systems to shape-memory alloys and self-healing coatings. The ability to programme such materials, however, is basic compared to the ability of a living organism to observe, understand and respond to its environment. Here we demonstrate the relocation of biological information processing systems from cells to materials. We achieved this by operating small, programmable genetic devices outside the confines of a living cell and inside hydrogel matrices. These results establish a method for developing materials functionally enhanced with molecular machinery from biological systems.

scholarly journals High throughput gene expression profiling of yeast colonies with microgel-culture Drop-seq

2018 ◽  
Author(s):  
Leqian Liu ◽  
Chiraj Dalal ◽  
Ben Heineike ◽  
Adam Abate
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Transcriptional Profiling ◽  
Cost Effective ◽  
Chemical Production ◽  
Efficient Test ◽  
Large Numbers ◽  
Design Build ◽  
Gene Expression Heterogeneity ◽  
High Throughput Gene Expression

AbstractYeasts can be engineered into “living foundries” for non-natural chemical production by reprogramming their genome using a synthetic biology “design-build-test” cycle. While methods for “design” and “build” are scalable and efficient, “test” remains a labor-intensive bottleneck, limiting the effectiveness of the genetic reprogramming results. Here we describe Isogenic Colony Sequencing (ICO-seq), a massively-parallel strategy to assess the gene expression, and thus engineered pathway efficacy, of large numbers of genetically distinct yeast colonies. We use the approach to characterize opaque-white switching in 658 C. albicans colonies. By profiling transcriptomes of 1642 engineered S. cerevisiae strains, we use it to assess gene expression heterogeneity in a protein mutagenesis library. Our approach will accelerate synthetic biology by allowing facile and cost-effective transcriptional profiling of large numbers of genetically distinct yeast strains.

scholarly journals Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Simone Balzer Le ◽  
Ingerid Onsager ◽  
Jon Andreas Lorentzen ◽  
Rahmi Lale
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Expression Profiles ◽  
Regulation Of Gene Expression ◽  
Design Concept ◽  
Fine Tuning ◽  
The Novel ◽  
Wide Range ◽  
Application Potential

Abstract Bacterial 5′ untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5′ UTR, dual UTR, utilizing the transcriptional and translational characteristics of 5′ UTRs in a single expression cassette. The dual UTR consists of two 5′ UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximization of protein production.

scholarly journals Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Thomas C. Williams ◽  
Xin Xu ◽  
Martin Ostrowski ◽  
Isak S. Pretorius ◽  
Ian T. Paulsen
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
High Throughput ◽  
Positive Feedback ◽  
High Throughput Screening ◽  
Transcriptional Regulator ◽  
Gfp Expression ◽  
Dynamic Regulation ◽  
Wide Range ◽  
Screening Efficiency

Biosensors are valuable and versatile tools in synthetic biology that are used to modulate gene expression in response to a wide range of stimuli. Ligand responsive transcription factors are a class of biosensor that can be used to couple intracellular metabolite concentration with gene expression to enable dynamic regulation and high-throughput metabolite producer screening. We have established the Saccharomyces cerevisiae WAR1 transcriptional regulator and PDR12 promoter as an organic acid biosensor that can be used to detect varying levels of para-hydroxybenzoic acid (PHBA) production from the shikimate pathway and output green fluorescent protein (GFP) expression in response. The dynamic range of GFP expression in response to PHBA was dramatically increased by engineering positive-feedback expression of the WAR1 transcriptional regulator from its target PDR12 promoter. In addition, the noise in GFP expression at the population-level was controlled by normalising GFP fluorescence to constitutively expressed mCherry fluorescence within each cell. These biosensor modifications increased the high-throughput screening efficiency of yeast cells engineered to produce PHBA by 5,000-fold, enabling accurate fluorescence activated cell sorting isolation of producer cells that were mixed at a ratio of 1 in 10,000 with non-producers. Positive-feedback, ratiometric transcriptional regulator expression is likely applicable to many other transcription-factor/promoter pairs used in synthetic biology and metabolic engineering for both dynamic regulation and high-throughput screening applications.

scholarly journals Robust gene expression control in human cells with a novel universal TetR aptamer splicing module

2019 ◽  
Vol 47 (20) ◽  
pp. e132-e132 ◽  
Author(s):  
Adam A Mol ◽  
Florian Groher ◽  
Britta Schreiber ◽  
Ciaran Rühmkorff ◽  
Beatrix Suess
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Dynamic Range ◽  
Intron Retention ◽  
Human Cells ◽  
Fine Tuning ◽  
Genomic Context ◽  
Mammalian Cell Lines ◽  
Gene Expression Control ◽  
Wide Range

Abstract Fine-tuning of gene expression is desirable for a wide range of applications in synthetic biology. In this context, RNA regulatory devices provide a powerful and highly functional tool. We developed a versatile, robust and reversible device to control gene expression by splicing regulation in human cells using an aptamer that is recognized by the Tet repressor TetR. Upon insertion in proximity to the 5′ splice site, intron retention can be controlled via the binding of TetR to the aptamer. Although we were able to demonstrate regulation for different introns, the genomic context had a major impact on regulation. In consequence, we advanced the aptamer to develop a splice device. Our novel device contains the aptamer integrated into a context of exonic and intronic sequences that create and maintain an environment allowing a reliable and robust splicing event. The exon-born, additional amino acids will then be cleaved off by a self-cleaving peptide. This design allows portability of the splicing device, which we confirmed by demonstrating its functionality in different gene contexts. Intriguingly, our splicing device shows a high dynamic range and low basal activity, i.e. desirable features that often prove a major challenge when implementing synthetic biology in mammalian cell lines.

scholarly journals Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques

Amino Acids ◽  
2021 ◽  
Author(s):  
Agnes Molnar ◽  
Tamas Lakat ◽  
Adam Hosszu ◽  
Beata Szebeni ◽  
Anna Balogh ◽  
...  
Keyword(s):  
Biological Samples ◽  
Basic Research ◽  
Cost Effective ◽  
Specific Reference ◽  
Metabolome Analysis ◽  
C Storage ◽  
Mouse Tissues ◽  
Freeze Dried ◽  
Wide Range ◽  
Similar Quantity

AbstractLyophilization is a cost-effective method for biological specimen preservation but detailed tissue-specific reference protocols are still lacking. Moreover, data are limited on the long-term stability of proteins and nucleic acids in lyophilized samples.Here, we offer lyophilization protocols for various rat and mouse tissues (kidney, heart, liver, lung, aorta, and skin) coupled with technical hints for optimal sample preparation. We demonstrate that lyophilized samples stored at 4 °C for 20 months can yield protein and RNA of similar quantity and quality to −80 °C storage, while phosphorylated proteins are preserved as well. Freeze-dried and subsequently pulverized samples can provide more consistent, more reliable data especially when investigating focal injuries, such as fibrosis. We developed a protocol for the concentration of biological solutions and achieved 20-times concentration in human peritoneal dialysis effluent solution which enables the previously unattainable detection of proteins in these samples. We established a method for water removal as well as accurate water content measurement of fecal samples, which can be valuable for gut metabolome analysis.Taken together, lyophilization is a valuable tool for the preservation of biological samples with many advantages. We aim to draw attention to the wide range of possibilities offered by freeze drying in pre-clinical or basic research.

scholarly journals High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

2018 ◽  
Author(s):  
Mandeep Singh ◽  
Ghamdan Al-Eryani ◽  
Shaun Carswell ◽  
James M. Ferguson ◽  
James Blackburn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
High Throughput ◽  
Clonal Evolution ◽  
Primary Tumour ◽  
Transcriptome Profiling ◽  
Cost Effective ◽  
Short Read ◽  
Wide Range ◽  
Long Read

AbstractHigh-throughput single-cell RNA-Sequencing is a powerful technique for gene expression profiling of complex and heterogeneous cellular populations such as the immune system. However, these methods only provide short-read sequence from one end of a cDNA template, making them poorly suited to the investigation of gene-regulatory events such as mRNA splicing, adaptive immune responses or somatic genome evolution. To address this challenge, we have developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We use Repertoire And Gene Expression sequencing (RAGE-seq) to accurately characterize full-length T cell (TCR) and B cell (BCR) receptor sequences and transcriptional profiles of more than 7,138 lymphocytes sampled from the primary tumour and draining lymph node of a breast cancer patient. With this method we show that somatic mutation, alternate splicing and clonal evolution of T and B lymphocytes can be tracked across these tissue compartments. Our results demonstrate that RAGE-Seq is an accessible and cost-effective method for high-throughput deep single cell profiling, applicable to a wide range of biological challenges.

Reprogramming bacteria with RNA regulators

2019 ◽  
Vol 47 (5) ◽  
pp. 1279-1289 ◽  
Author(s):  
Patrícia Apura ◽  
Susana Domingues ◽  
Sandra C. Viegas ◽  
Cecília M. Arraiano
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Building Blocks ◽  
Natural Mode ◽  
Cellular Functions ◽  
Diverse Range ◽  
Control Of Gene Expression ◽  
Precise Control ◽  
Engineering Discipline ◽  
Industrial Needs

Abstract The revolution of genomics and growth of systems biology urged the creation of synthetic biology, an engineering discipline aiming at recreating and reprogramming cellular functions for industrial needs. There has been a huge effort in synthetic biology to develop versatile and programmable genetic regulators that would enable the precise control of gene expression. Synthetic RNA components have emerged as a solution, offering a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their compactness, structure and way of action, several types of RNA devices that act on DNA, RNA and protein have been characterized and applied in synthetic biology. RNA-based approaches are more ‘economical' for the cell, since they are generally not translated. These RNA-based strategies act on a much shorter time scale than transcription-based ones and can be more efficient than protein-based mechanisms. In this review, we explore these RNA components as building blocks in the RNA synthetic biology field, first by explaining their natural mode of action and secondly discussing how these RNA components have been exploited to rewire bacterial regulatory circuitry.

scholarly journals Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

2017 ◽  
Vol 6 (12) ◽  
pp. 2198-2208 ◽  
Author(s):  
Andriy Didovyk ◽  
Taishi Tonooka ◽  
Lev Tsimring ◽  
Jeff Hasty
Keyword(s):  
Gene Expression ◽  
Free Gene ◽  
Bacterial Lysates
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