9 research outputs found
Multicolor plate reader fluorescence calibration
Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells
Cloning whole bacterial genomes in yeast
Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation
Single-cell measurement of plasmid copy number and promoter activity
Accurate measurements of promoter activities are crucial for predictably building genetic systems. Here we report a method to simultaneously count plasmid DNA, RNA transcripts, and protein expression in single living bacteria. From these data, the activity of a promoter in units of RNAP/s can be inferred. This work facilitates the reporting of promoters in absolute units, the variability in their activity across a population, and their quantitative toll on cellular resources, all of which provide critical insights for cellular engineering
The genotypeāphenotype landscape of an allosteric protein
Abstract Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotypeāphenotype relationships remains elusive. Here, we report the largeāscale measurement of the genotypeāphenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines longāread and shortāread DNA sequencing, we quantitatively measure the doseāresponse curves for nearly 105 variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequenceāstructureāfunction relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neuralānetwork models, but unpredictable changes also occur. For example, we were surprised to discover a new bandāstop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions
Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases
Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination
A pesar de que la arquitectura actual parece tener una tendencia general hacia lo mediĆ”tico, aproximĆ”ndose cada vez mĆ”s a convertirse en un producto de consumo del mercado del arte, aĆŗn existen unos pocos arquitectos que conservan unos valores y una forma de pensar y hacer arquitectura considerados ya por muchos obsoletos. Uno de estos arquitectos es el madrileƱo VĆctor LĆ³pez Cotelo y con este trabajo se pretende estudiar su obra para tratar de mostrar no sĆ³lo que no se trata de valores obsoletos, sino que suponen el mejor camino para llegar a la verdadera arquitectura y por ello estarĆ”n siempre vigentes, sin importar los cambios que haya en las modas. Para estudiar la obra de este arquitecto se realiza un recorrido por tres de sus edificios, situados en la ribera del rĆo Sarela, en Santiago de Compostela. Tres obras (Ponte Sarela, la VaquerĆa y PontepedriƱa) que parten de ruinas de piedra y variadas topografĆas para llegar a tres magnĆficos edificios en los que el tiempo es el protagonista, el pasado y el presente se dan la mano y la arquitectura se pone al servicio de la vida, con el objetivo de mejorar la de quienes la habitan
Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination
ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes