For instance, the oriVs of pSC101 and RK2 carry typical short direct repeats for binding of a plasmid-encoded single Rep protein, while RSF1010 carries no less than three rep genes encoding proteins for priming, unwinding of DNA, and initiation of replication ( repA, repB, repC. The genetic structure and mode of replication heavily differs between replication systems. The remaining eight replication systems are either broad host range systems (2, RK2 3, pBBR1 4, pRO1600/ColE1 5, RSF1010) or specific for enteric bacteria (6, p15A 7, pSC101 8, pUC 9, pBR322). The first one, the origin of the R6K plasmid, is intended for suicide vectors and requires the π protein for plasmid maintenance usually provided by an appropriate pir+ host strain. The SEVA platform currently contains nine different replication systems. In this study, we chose the SEVA standard as underlying architecture for plasmid design, as this repository provides a coherent modular plasmid structure and a wealth of replication systems that can be used instantly. the biobricks standard accompanied by its Registry of Standard Biological Parts or the Standard European Vector Architecture (SEVA) for systematic assembly of plasmids. Nevertheless, there is a demand for standardized genetic parts with predictable function, and recently, efforts were undertaken to create platforms for systematic creation, annotation, and combination of such parts. However, only sparse information regarding PCN and expression heterogeneity is available for the wealth of different replication systems used in laboratories world-wide. shown by Kittleson and colleagues using a library of 20 rep mutants. As plasmids follow a discrete distribution, a low mean PCN may also lead to higher cell to cell heterogeneity regarding PCN and gene expression, as e.g. An example of heterogeneity caused by loss of a very low-copy plasmid was the bimodal distribution of EGFP fluorescence in Pseudomonas putida. This reduces heterogeneity, however, no such system was included in this study. It must be noted that this behavior is different in low-copy plasmids that carry active partitioning systems to ensure faithful distribution of one or few plasmid copies from mother to daughter cells. A low mean PCN furthermore promotes failure of plasmid distribution to daughter cells, while a higher mean PCN is supposed to ensure that every daughter obtains plasmid molecules. įor biotechnological applications it is highly desirable to know the range of copy numbers that can be expected from a particular vector, as the gene dosage can be crucial for efficient protein production. The essential part of a plasmid primarily determining its copy number (PCN) is the replication system, in most cases composed of a vegetative origin of replication ( oriV), and a gene encoding the replication initiation protein ( rep).
Those parts include antibiotic resistance cassettes, replication and induction systems, and a great number of ‘cargo’ genes. The result is an overwhelming number of plasmid parts which are often poorly characterized. However, the design and cloning of plasmid vectors in many laboratories world-wide did not follow any systematic rules. Plasmids replicate autonomously from the bacterial chromosome and are usually present in more than one copy per cell, leading to higher recombinant gene dosage. Plasmids have been used in biotechnology for decades as they are easy to manipulate and transfer to host cells. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, respectively.
The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. The average PCN for all constructs was determined by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. In this study, the PCN was determined in a set of seven SEVA-based expression plasmids only differing in the replication system.