Single-cell measurement of plasmid copy number and promoter activity.
Ontology highlight
ABSTRACT:
SUBMITTER: Shao B
PROVIDER: S-EPMC7935883 | biostudies-literature | 2021 Mar
REPOSITORIES: biostudies-literature
Publications
Single-cell measurement of plasmid copy number and promoter activity.
Shao Bin B Rammohan Jayan J Anderson Daniel A DA Alperovich Nina N Ross David D Voigt Christopher A CA
Nature communications 20210305 1
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 provid ...[more]
Similar Datasets
Project description:Bacterial high-copy-number (hcn) plasmids provide an excellent model to study the underlying physical mechanisms of DNA segment segregation in an intracellular context. Using two-color fluorescent repressor-operator systems and a synthetic repressible replication origin, we tracked the motion and segregation of single hcn plasmid molecules in individual cells. The plasmid diffusion dynamics revealed between-plasmid temporal associations (clustering) as well as entropic and elastic recoiling forces in the confined intracellular spaces outside of nucleoids. These two effects could be effectively used in models to predict the heterogeneity of segregation. Additionally, the motile behaviors of hcn plasmids provide quantitative estimates of entropic exclusion strength and dynamic associations between DNA segments. Overall, this study utilizes a, to our knowledge, novel approach to predict the polymer dynamics of DNA segments in spatially confined, crowded cellular compartments as well as during bacterial chromosome segregation.
| S-EPMC6400962 | biostudies-literature
Project description:Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.
| S-EPMC5234771 | biostudies-literature
Project description:Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data.
| S-EPMC3245619 | biostudies-literature
Project description:Plasmids are one of the most commonly used platforms for genetic engineering and recombinant gene expression in bacteria. The range of available copy numbers for cloning vectors is largely restricted to the handful of Origins of Replication (ORIs) that have been isolated from plasmids found in nature. Here, we introduce two systems that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell: a plasmid with an anhydrotetracycline-controlled copy number, and a parallelized assay that is used to generate a continuous spectrum of 1194 ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our systems enable the precise control of gene expression, and our results highlight the importance of tuning plasmid copy number as a powerful tool for the optimization of synthetic biological systems.
| S-EPMC9263177 | biostudies-literature
Project description:Currently, there is a lack of software for detecting copy number variations and constructing copy number profile for the whole genome from single-cell DNA sequencing data, which are often of low coverage and high technical noises. Here we introduce a new toolkit, SCNV, which features an efficient bin-free segmentation approach and provides the highest resolution possible for breakpoint detection and the subsequent copy number calling. SCNV can auto-tune parameters based on a set of normal cells from the same batch to adjust for the technical noise level of the data, facilitating its application to data gathered from different platforms and different studies.
| S-EPMC6171490 | biostudies-literature
Project description:To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method.
| S-EPMC8708932 | biostudies-literature
Project description:MotivationCopy number alterations are driving forces of tumour development and the emergence of intra-tumour heterogeneity. A comprehensive picture of these genomic aberrations is therefore essential for the development of personalised and precise cancer diagnostics and therapies. Single-cell sequencing offers the highest resolution for copy number profiling down to the level of individual cells. Recent high-throughput protocols allow for the processing of hundreds of cells through shallow whole-genome DNA sequencing. The resulting low read-depth data poses substantial statistical and computational challenges to the identification of copy number alterations.ResultsWe developed SCICoNE, a statistical model and MCMC algorithm tailored to single-cell copy number profiling from shallow whole-genome DNA sequencing data. SCICoNE reconstructs the history of copy number events in the tumour and uses these evolutionary relationships to identify the copy number profiles of the individual cells. We show the accuracy of this approach in evaluations on simulated data and demonstrate its practicability in applications to two breast cancer samples from different sequencing protocols.Availability and implementationSCICoNE is available at https://github.com/cbg-ethz/SCICoNE.
| S-EPMC11897432 | biostudies-literature
Project description:Natural microbial populations exploit phenotypic heterogeneity for survival and adaptation. However, in engineering biology, limiting the sources of variability is a major focus. Here we show that intentionally coupling distinct plasmids via shared replication mechanisms enables bacterial populations to adapt to their environment. We demonstrate that plasmid coupling of carbon-metabolizing operons facilitates copy number tuning of an essential but burdensome construct through the action of a stably maintained, non-essential plasmid. For specific cost-benefit situations, incompatible two-plasmid systems can stably persist longer than compatible ones. We also show using microfluidics that plasmid coupling of synthetic constructs generates population-state memory of previous environmental adaptation without additional regulatory control. This work should help to improve the design of synthetic populations by enabling adaptive engineered strains to function under changing growth conditions without strict fine-tuning of the genetic circuitry.
| S-EPMC11623956 | biostudies-literature
Project description:Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.
| S-EPMC7848731 | biostudies-literature
Project description:Uniform amplification of low input DNA is important for applications across biology, including single-cell genomics, forensic science, and microbial and viral sequencing. However, the requisite biochemical amplification methods are prone to bias, skewing sequence proportions and obscuring signals relating to copy number. Digital droplet multiple displacement amplification enables uniform amplification, but requires expert knowledge of microfluidics to generate monodisperse emulsions. In addition, existing microfluidic methods are tedious and labor intensive for preparing many samples. Here, we introduce rapid emulsification multiple displacement amplification, a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter. While conventional microfluidic devices require >10 minutes to emulsify a sample, our system takes tens of seconds and yields data of equivalent quality. We demonstrate the approach by using it to accurately measure copy number variation in single cancer cells.
| S-EPMC6108428 | biostudies-literature