plasmid copy number
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Author(s):  
Claudia Igler ◽  
Jana S. Huisman ◽  
Berit Siedentop ◽  
Sebastian Bonhoeffer ◽  
Sonja Lehtinen

As infectious agents of bacteria and vehicles of horizontal gene transfer, plasmids play a key role in bacterial ecology and evolution. Plasmid dynamics are shaped not only by plasmid–host interactions but also by ecological interactions between plasmid variants. These interactions are complex: plasmids can co-infect the same cell and the consequences for the co-resident plasmid can be either beneficial or detrimental. Many of the biological processes that govern plasmid co-infection—from systems that exclude infection by other plasmids to interactions in the regulation of plasmid copy number—are well characterized at a mechanistic level. Modelling plays a central role in translating such mechanistic insights into predictions about plasmid dynamics and the impact of these dynamics on bacterial evolution. Theoretical work in evolutionary epidemiology has shown that formulating models of co-infection is not trivial, as some modelling choices can introduce unintended ecological assumptions. Here, we review how the biological processes that govern co-infection can be represented in a mathematical model, discuss potential modelling pitfalls, and analyse this model to provide general insights into how co-infection impacts ecological and evolutionary outcomes. In particular, we demonstrate how beneficial and detrimental effects of co-infection give rise to frequency-dependent selection on plasmid variants. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.


2021 ◽  
Author(s):  
Santiago Chaillou ◽  
Eleftheria-Pinelopi Stamou ◽  
Leticia L. Torres ◽  
Ana B. Riesco ◽  
Warren Hazelton ◽  
...  

Plasmids of the ColE1 family are among the most frequently used plasmids in molecular biology. They were adopted early in the field for many biotechnology applications, and as model systems to study plasmid biology. The mechanism of replication of ColE1 plasmids is well understood, involving the interaction between a plasmid-encoded sense-antisense gene pair (RNAI and RNAII). Because of its mechanism of replication, bacterial cells cannot maintain two different plasmids with the same origin, with one being rapidly lost from the population — a process known as plasmid incompatibility. While mutations in the regulatory genes RNAI and RNAII have been reported to make colE1 plasmids more compatible, there has been no attempt to engineer compatible colE1 origins, which can be used for multi-plasmid applications and that can bypass design constrains created by the current limited plasmid origin repertoire available. Here, we show that by targeting sequence diversity to the loop regions of RNAI (and RNAII), it is possible to select new viable colE1 origins that are compatible with the wild-type one. We demonstrate origin compatibility is not simply determined by sequence divergence in the loops, and that pairwise compatibility is not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality. New characterised plasmid origins increase flexibility and accessible complexity of design for challenging synthetic biology applications where biological circuits can be dispersed between multiple independent genetic elements.


2021 ◽  
Vol 22 (16) ◽  
pp. 8538
Author(s):  
Andrés Romero ◽  
Vicente Rojas ◽  
Verónica Delgado ◽  
Francisco Salinas ◽  
Luis F. Larrondo

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light–oxygen–voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call “HAP-LOV”, displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.


Author(s):  
Benjamin David ◽  
Jinbei Li ◽  
Faisal Masood ◽  
Caroline Blassick ◽  
Paul Jensen ◽  
...  

Quantitative PCR (qPCR) has numerous applications in biology. In an education setting, qPCR provides students an opportunity to better understand the PCR mechanism by providing both quantitative information about the reactions and also data to troubleshoot PCRs (e.g., melt curves). Here, we present a relatively short (2-h) laboratory activity to demonstrate qPCR to quantify plasmid copy number (CN) by measuring the cycle threshold ( C T ) values for a genomic gene and a plasmid gene using transformed cells as a template. The activity can be combined with additional laboratory exercises, including bacterial transformation, to create the template to be used in the qPCRs. This lab activity is ideal for undergraduate laboratory courses that include recombinant DNA technology.


2021 ◽  
Author(s):  
Miles V Rouches ◽  
Yasu Xu ◽  
Louis Cortes ◽  
Guillaume Lambert

Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of 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 massively parallel 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 plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as tool for the optimization of synthetic biological systems.


Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 776
Author(s):  
Samuel Lihan ◽  
Sai Y. Lee ◽  
Seng C. Toh ◽  
Sui S. Leong

Background: The emergence of plasmid-mediated antibiotic resistance in Escherichia coli in water resources could pose a serious threat to public health. The study aims to investigate the dispersion of plasmid-mediated antibiotic-resistant E. coli from six rivers in Sarawak and two aquaculture farms in Borneo. Methods: A total of 74 water samples were collected for the determination of their bacteria colony count. An IMViC test identified 31 E. coli isolates and tested their susceptibility against twelve clinically important antibiotics. The extraction of plasmid DNA was done using alkali lysis SDS procedures. Characteristics, including plasmid copy number, molecular weight size, resistance rate and multiple antibiotic resistance (MAR), were assessed. Results: Our findings revealed that bacterial counts in rivers and aquaculture farms ranged from log 2.00 to 3.68 CFU/mL and log 1.70 to 5.48 cfu/mL, respectively. Resistance to piperacillin (100%) was observed in all E. coli; resistance to amoxicillin (100%) and ampicillin (100%) was observed in E. coli found in aquaculture farms; resistance to streptomycin (93%) was observed in E. coli found in rivers. All E. coli were resistant to ≥2 antibiotics and formed 26 MAR profiles, ranging from an index of 0.17 to 0.83, indicating that there are high risks of contamination. Some (48.4%) of the E. coli were detected with plasmids (1.2 to >10 kb), whereas 51.6% of the E. coli did not harbor any plasmids. The plasmid copy numbers reported were one plasmid (n = 7), two plasmids (n = 4), ≥ two plasmids (4). E. coli isolated from the Muara Tuang River showed the highest-molecular-weight plasmids. A statistical analysis revealed that there is no significant correlation (r = 0.21, p = 0.253) between the number of plasmids and the MAR index of the tested isolates. Conclusion: The distribution of MAR in E. coli from rivers is higher compared to the aquaculture environment. Our study suggests that MAR in isolates could be chromosome-mediated. Our results suggest that riverbed sediments could serve as reservoirs for MAR bacteria, including pathogens, under different climatic conditions, and their analysis could provide information for public health concerns.


2021 ◽  
Author(s):  
Claudia Igler ◽  
Jana Sanne Huisman ◽  
Berit Siedentop ◽  
Sebastian Bonhoeffer ◽  
Sonja Lehtinen

As infectious agents of bacteria and vehicles of horizontal gene transfer, plasmids play a key role in bacterial ecology and evolution. Plasmid dynamics are shaped not only by plasmid-host interactions, but also by ecological interactions between plasmid variants. These interactions are complex: plasmids can co-infect the same host cell and the consequences for the co-resident plasmid can be either beneficial or detrimental. Many of the biological processes that govern plasmid co-infection--from systems to exclude infection by other plasmids to interactions in the regulation of plasmid copy number per cell--are well characterised at a mechanistic level. Modelling plays a central role in translating such mechanistic insights into predictions about plasmid dynamics, and in turn, the impact of these dynamics on bacterial evolution. Theoretical work in evolutionary epidemiology has shown that formulating models of co-infection is not trivial, as some modelling choices can introduce unintended ecological assumptions. Here, we review how the biological processes that govern co-infection can be represented in a mathematical model, discuss potential modelling pitfalls, and analyse this model to provide general insights into how co-infection impacts eco-evolutionary outcomes. In particular, we demonstrate how beneficial and detrimental effects of co-infection give rise to frequency-dependent selection.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bin Shao ◽  
Jayan Rammohan ◽  
Daniel A. Anderson ◽  
Nina Alperovich ◽  
David Ross ◽  
...  

AbstractAccurate 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.


2021 ◽  
Vol 12 ◽  
Author(s):  
Bo-Xuan Zeng ◽  
Ming-Dong Yao ◽  
Wen-Hai Xiao ◽  
Yun-Zi Luo ◽  
Ying Wang ◽  
...  

In Saccharomyces cerevisiae, conventional 2μ-plasmid based plasmid (pC2μ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2μ plasmid (pE2μ) brings the issues concerning plasmid stability and copy number of pC2μ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2μ and built the pE2μ multi-copy system by insertion of the target DNA element and elimination of the original pE2μ plasmid. The resulting plasmid pE2μRAF1 and pE2μREP2 demonstrated higher copy number and slower loss rate than a pC2μ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2μRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2μ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2μ multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2μRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2μ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2μ due to the more stable pE2μ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.


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