RESUMO
Metabolic engineering has expanded from a focus on designs requiring a small number of genetic modifications to increasingly complex designs driven by advances in genome-scale engineering technologies. Metabolic engineering has been generally defined by the use of iterative cycles of rational genome modifications, strain analysis and characterization, and a synthesis step that fuels additional hypothesis generation. This cycle mirrors the Design-Build-Test-Learn cycle followed throughout various engineering fields that has recently become a defining aspect of synthetic biology. This review will attempt to summarize recent genome-scale design, build, test, and learn technologies and relate their use to a range of metabolic engineering applications.
Assuntos
Genoma/genética , Engenharia Metabólica/métodos , Biologia Sintética/tendências , Animais , DNA/genética , HumanosRESUMO
Performance and utility of microfluidic systems are often overshadowed by the difficulties and costs associated with operation and control. As a step toward the development of a more efficient platform for microfluidic control, we present a distributed pressure generation scheme whereby independently tunable pressure sources can be simultaneously controlled by using a single acoustic source. We demonstrate how this scheme can be used to perform precise droplet positioning as well as merging, splitting, and sorting within open microfluidic networks. We further show how this scheme can be implemented for control of continuous-flow systems, specifically for generation of acoustically tunable liquid gradients. Device operation hinges on a resonance-decoding and rectification mechanism by which the frequency content in a composite acoustic input is decomposed into multiple independently buffered output pressures. The device consists of a bank of 4 uniquely tuned resonance cavities (404, 484, 532, and 654 Hz), each being responsible for the actuation of a single droplet, 4 identical flow-rectification structures, and a single acoustic source. Cavities selectively amplify resonant tones in the input signal, resulting in highly elevated local cavity pressures. Fluidic-rectification structures then serve to convert the elevated oscillating cavity pressures into unidirectional flows. The resulting pressure gradients, which are used to manipulate fluids in a microdevice, are tunable over a range of approximately 0-200 Pa with a control resolution of 10 Pa.
Assuntos
Técnicas Analíticas Microfluídicas , Acústica , PressãoRESUMO
Procedures requiring precise and accurate positioning of particles and cells have impacted a broad range of research interests including molecular detection, self-assembly and tissue and cell engineering. These fields would be greatly aided by more advanced, yet straightforward, micro-object positioning methods that are precise, scalable, responsive and flexible. We have developed an arrayed, multilayer surface patterned microfluidic device which uses laminar convective flow to actively position particles into any desired, two-dimensional, predesigned pattern. Objects including 10 microm polystyrene particles and Saccharomycodes ludwigii cells are rapidly (approximately 2 s) loaded onto vacuum-actuated holes, allowing us to both generate anisotropic particles and culture S. ludwigii cells. The device was further modified to individually control two sets of holes, adding control of pattern composition. With rapid, precise and adaptable operation, multilayer microfluidic devices should greatly assist in research where precise object placement and proximity is necessary.
Assuntos
Biopolímeros/química , Biopolímeros/isolamento & purificação , Técnicas de Cultura de Células/instrumentação , Separação Celular/instrumentação , Análise em Microsséries/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Micromanipulação/instrumentação , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
Particles combining multiple anisotropy dimensions offer possibilities for self-assembly that have not been extensively explored to date. The scope for assembly of microparticles in which the anisotropy dimensions of internal bond angle and chemical ordering have been varied is investigated. Colloidal assemblies with interesting open (i.e., non-close-packed) structures can be assembled from these building blocks. The structure of the assemblies formed is linked to the building block anisotropy because the steric constraints introduced induce deviations from close packing. Key challenges addressed in pursuit of these structures are parallelization of microfluidic synthesis methods, simulation to efficiently search the available anisotropy space, and methods that characterize the properties of the resulting assemblies. This combined program of synthesis, simulation, assembly, and characterization may be applied to develop design rules that guide efforts to fabricate microparticle building blocks and their assemblies.
RESUMO
Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites. This approach works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This approach, called barcoded-TRACE (bTRACE) was applied to assess E. coli populations engineered by recursive multiplex recombineering across both 6-target sites and 31-target sites. The 31-target library was then tracked throughout growth selections in the presence and absence of isopentenol (a potential next-generation biofuel). We also use the resolution of bTRACE to compare the influence of technical and biological noise on genome engineering efforts.
Assuntos
Algoritmos , Engenharia Genética , Escherichia coli/genética , Biblioteca Gênica , Genoma Bacteriano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Plasmídeos/genética , Plasmídeos/metabolismo , Análise de Sequência de DNARESUMO
Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.
Assuntos
Mapeamento Cromossômico/métodos , Análise Mutacional de DNA/métodos , Edição de Genes/métodos , Engenharia Metabólica/métodos , Polimorfismo de Nucleotídeo Único/genética , Engenharia de Proteínas/métodos , Algoritmos , Genoma Bacteriano/genética , Genoma Fúngico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Metaboloma/genética , Nucleotídeos/genética , Proteoma/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SoftwareRESUMO
Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library. These redundancies increase library size and downstream screening efforts. Here, we present a dynamic approach to compress codons for any desired list of amino acids, taking into account codon usage. This results in a unique codon collection for every amino acid to be mutated, with the desired redundancy level. Finally, we demonstrate that this approach can be used to design precise oligo libraries amendable to recombineering and CRISPR-based genome editing to obtain a diverse population with high efficiency.
Assuntos
Códon/genética , Mutagênese/genética , Algoritmos , Aminoácidos/genética , Biblioteca Gênica , Mutação/genética , Oligonucleotídeos/genética , Engenharia de Proteínas/métodosRESUMO
Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies. We used TRACE to map growth selection dynamics for Escherichia coli combinatorial libraries created by recursive multiplex recombineering at a depth 10(4)-fold greater than before. TRACE was used to identify genotype-to-phenotype correlations and to map the evolutionary trajectory of two individual combinatorial mutants in E. coli. Combinatorial mutations in the human ES2 ovarian carcinoma cell line were also assessed with TRACE. TRACE completes the combinatorial engineering cycle and enables more sophisticated approaches to genome engineering in both bacteria and eukaryotic cells than are currently possible.
Assuntos
Escherichia coli/genética , Engenharia Genética , Variação Genética , Mutação/genética , Estudos de Associação Genética , Genoma Bacteriano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Célula ÚnicaRESUMO
Nanolitre droplets in microfluidic devices can be used to perform thousands of independent chemical and biological experiments while minimizing reagents, cost and time. However, the absence of simple and versatile methods capable of controlling the contents of these nanolitre chemical systems limits their scientific potential. To address this, we have developed a method that is simple to fabricate and can continuously control nanolitre chemical systems by integrating a time-resolved convective flow signal across a permeable membrane wall. With this method, we can independently control the volume and concentration of nanolitre-sized drops without ever directly contacting the fluid. Transport occurring in these systems was also analyzed and thoroughly characterized. We achieved volumetric fluid introduction and removal rates ranging from 0.23 to 4.0 pL s(-1). Furthermore, we expanded this method to perform chemical processes. We precipitated silver chloride using a flow signal of sodium chloride and silver nitrate droplets. From there, we were able to separate sodium chloride reactants with a water flow signal, and dissolve silver chloride solids with an ammonia hydroxide flow signal. Finally, we demonstrate the potential to deliver large molecules and perform physical processes like crystallization and particle packing.
Assuntos
Técnicas Analíticas Microfluídicas , Nanotecnologia/métodos , Hidróxido de Amônia , Dimetilpolisiloxanos/química , Hidróxidos/química , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Nanotecnologia/instrumentação , Óleos/química , Compostos de Prata/química , Nitrato de Prata/química , Cloreto de Sódio/química , Tensoativos/química , Temperatura , Água/químicaRESUMO
Pressure-driven flow control systems are a critical component in many microfluidic devices. Compartmentalization of this functionality into a stand-alone module possessing a simple interface would allow reduction of the number of pneumatic interconnects required for fluidic control. Ideally, such a module would also be sufficiently compact for implementation in portable platforms. In our current work, we show the feasibility of using a modular array of Venturi pressure microregulators for coordinated droplet manipulation. The arrayed microregulators share a single pressure input and are capable of outputting electronically controlled pressures that can be independently set between ±1.3 kPa. Because the Venturi microregulator operates by thermal perturbation of a choked gas flow, this output range corresponds to a temperature variation between 20 and 95°C. Using the array, we demonstrate loading, splitting, merging, and independent movement of multiple droplets in a valveless microchannel network.
RESUMO
We have observed the non-uniform distribution of DNA molecules during PAGE in a microfabricated system. Confocal laser scanning microscopy was used to visualize fluorescently labeled DNA during electrophoretic migration. The distribution of double-stranded DNA larger than 100 bp is observed to transition from a center-biased motion on the transverse plane 1 cm downstream from injection to an edge-biased motion 2 cm downstream. Although this distribution increased with increasing dsDNA size in a cross-linked gel, no similar distribution was found with the same dsDNA molecules in a linear polyacrylamide solution (6%). Simulations of DNA distribution in gels suggest that DNA distribution non-uniformities may be caused by biased electrophoretic migration resulting from motion in an inhomogeneous gel system.