0000000001047053
AUTHOR
Roser Montagud-martínez
CRISPR-mediated strand displacement logic circuits with toehold-free DNA
DNA nanotechnology, and DNA computing in particular, has grown extensively over the past decade to end with a variety of functional stable structures and dynamic circuits. However, the use as designer elements of regular DNA pieces, perfectly complementary double strands, has remained elusive. Here, we report the exploitation of CRISPR-Cas systems to engineer logic circuits based on isothermal strand displacement that perform with toehold-free double-stranded DNA. We designed and implemented molecular converters for signal detection and amplification, showing good interoperability between enzymatic and nonenzymatic processes. Overall, these results contribute to enlarge the repertoire of su…
Additional file 2 of Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time
Additional file 2: Fig. S2. Evolutionary stability of a control system (E. coli transformed with plasmid pRHA27). Continuous fluorescence monitoring of four biological replicates (A-D) over one week.
CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation
The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was t…
Additional file 1 of Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time
Additional file 1: Fig. S1. Environmental robustness assessment of a control system (E. coli transformed with plasmid pRHA27; this system just expresses sfGFP under the control of the lac promoter). Fluorescence monitoring (normalized green fluorescence) when the culture medium changes (pH and temperature variation). Error bars correspond to standard errors over four biological replicates.
Additional file 3 of Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time
Additional file 3: Fig. S3. Schematics of the plasmids pRHA40, pRHA12, and pRHA1240.
Fitness Trade-Offs Determine the Role of the Molecular Chaperonin GroEL in Buffering Mutations
Molecular chaperones fold many proteins and their mutated versions in a cell and can sometimes buffer the phenotypic effect of mutations that affect protein folding. Unanswered questions about this buffering include the nature of its mechanism, its influence on the genetic variation of a population, the fitness trade-offs constraining this mechanism, and its role in expediting evolution. Answering these questions is fundamental to understand the contribution of buffering to increase genetic variation and ecological diversification. Here, we performed experimental evolution, genome resequencing, and computational analyses to determine the trade-offs and evolutionary trajectories of Escherich…
Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time
Synthetic biology aims at engineering gene regulatory circuits to end with cells (re)programmed on purpose to implement novel functions or discover natural behaviors. However, one overlooked question is whether the resulting circuits perform as intended in variety of environments or with time. Here, we considered a recently engineered genetic system that allows programming the cell to work as a minimal computer (arithmetic logic unit) in order to analyze its operability regime. This system involves transcriptional and post-transcriptional regulations. In particular, we studied the analog behavior of the system, the effect of physicochemical changes in the environment, the impact on cell gro…
Additional file 4 of Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time
Additional file 4: Dataset S1. Experimental and theoretical data of the half adder behavior. The Excel file contains an implementation of the mathematical model.