A recombinant yeast strain, saccharomyces cerevisiae dby746, containing the plasmid pjg317, was grown in a variety of fermentation modes including batch. Since both bla and dxs are singlecopy genes of pbr322 and e. Medium copy number expression vector and autoinducible promoter could be a solution for producing recombinant therapeutic proteins in industrial scale regarding plasmid stability, cost, and. The copy number and stability of several plasmid vectors in clostridium acetobutylicum atcc 824 were determined. Recombinant protein production in escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. Natural plasmids are generally large and maintained at low copy number and thus prone to be lost.
Related factors are plasmid copy number, structural plasmid stability and segregational plasmid stability. Copy number refers to the number of copies of a plasmid present in a cell. Transcription initiation of the copynumber control and betterthanrandom segregation genes of the broadhostrange and lowcopynumber plasmid psm19035 are subjected to repression by the autoregulated psm19035encoded. Although the newcomer likely knows that a plasmid is a small circular piece of dna found in bacterial cells, she may. Regarding to those facts, low copy number plasmids are more suitable to be used as expression vector in industrial scale, due to its high stability. Emergence of plasmid stability under nonselective conditions. Plasmid stability analysis based on a new theoretical. Plasmid pjg317 is quite unstable under nonselective conditions and its copy number and stability are influenced by both growth rate and nutrient supply by immobilising cells in calcium alginate gel beads, the plasmid could be stabilised and high volumetric productivities of up to 38 umlh attained. In this work we show that the absence of raf1 can affect both the plasmid stability and the steady sate copy number.
Introduction to addgenes resource any newcomer who joins a molecular biology lab will undoubtedly be asked to design, modify, or construct a plasmid. In fact, plasmid copy number can be influenced to some extent by all of the parameters tested. Plasmids represent a significant metabolic burden for their hosts 8. The plasmid copy number of pbr322 was then calculated by dividing the copy number of bla by the copy number of dxs. The studies performed should provide information on the stability of the gene copy number and any nucleotide sequence changes which have occurred in the expression. Guidance for industry food and drug administration. Friehs and others published plasmid copy number and plasmid stability find, read and cite all the research you need on. This near chromosomelike stability is due to two plasmid borne systems, namely the partitioning and the amplification systems comprising of the four proteins rep1, rep2, raf1 and. In addition to plasmid copy number, we investigated the effect of different sequences of. Plasmid copy number and disease severity in naturally.
Plasmid copy number and stability the plasmid copy number was determined by incorporation of methyl3hthymidine into total cellular dna, followed by the separation of chro mosomal and plasmid dna fractions by agarose gel electrophoresis, band excision, and measure ment of radiolabeled dna. Genetic stability testing for cell lines all genetic sequences are susceptible to random mutation. A major limitation to improving smallmolecule pharmaceutical production in streptomycetes is the inability of high copy number plasmids to tolerate large biosynthetic gene cluster inserts. Dec 19, 2016 plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Characterization of plasmid burden and copy number in. A sequence that affects the copy number and stability of. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this.
Structural stability describes the cor rect replication during cultivation of plasmid bearing cells leading to a nonal tered base sequence of the plasmid. The protocols were modified from the traditional ones to overcome the problems associated with unusual behavior of c. Plasmid copy number varies widely depending on the plasmid, ranging from as few as 12 copies per cell for f or r1 plasmids to up to 200 copies per cell for cole1 plasmid derivatives used for recombinant gene expression. Constant modification has brought forth countless plasmid vectors whose characteristics in terms of average plasmid copy number pcn and stability are rarely known. Segregational drift and the interplay between plasmid copy. Essential for plasmid monomerization and stability. Here, we present a simple theoretical model to study plasmid stability. Plasmid copy number was also determined in the ebs of six other lymphogranuloma venereum. To determine the plasmid copy number, two set of primers were used. Plasmid copy number mutation in repa gene encoding repa.
Escherichia coli plasmid copy number assay introduction for the plasmid copy number assay, two pairs of probes are required, one pair for the plasmid determination and one pair for the chromosome determination. The bottom control level encodes a single genetic circuit in a plasmid vector. Normally, the bacterium only tolerates one copy of the plasmid, which provides the molecular machinery for injecting toxins into host cells. Yb886 and yb886vstrains bearing plasmid prc1 were grown to the middle exponential phase, and plasmid dna from 5ml cultures od650 5 0. The monte carlo approach was used to analyze random fluctuations affecting plasmid replication and segregation leading to gradual reduction in the plasmid population within the host cell. This work is a theoretical analysis of random fluctuations and regulatory efficiency in genetic networks. The essential part of a plasmid primarily determining its copy number. Transcription initiation of the copy number control and betterthanrandom segregation genes of the broadhostrange and low copy number plasmid psm19035 are subjected to repression by the autoregulated psm19035encoded. The model predictions of the copy number of plasmids with the co1e1 origin of replication carrying a variety of regulatory mutations is very close to that observed experimentally.
Plasmid copy number varies widely depending on the plasmid, ranging from as few as 12 copies per cell for f or r1 plasmids 6 to up to 200 copies per cell for cole1 plasmid derivatives used for recombinant gene expression 7. Plasmids are typically present in multiple copies, and the copy number is relatively stable. Strategies for improving plasmid stability in genetically. Cole1type plasmids this probability is given by p0 21n, where n plasmid copy number in the. The crucial factor determining the pcn is the replication system. Us4935350a materials and methods for controlling plasmid. Structural plasmid stability exists, when all generated plasmids have the correct. The size of an expression plasmid is determined by restriction endonuclease digestion followed by gel electrophoresis. Effect of plasmid copy number and operator sequence on.
A recombinant yeast strain, saccharomyces cerevisiae dby746, containing the. The plasmids of chlamydia trachomatis and chlamydophila. The flp recombinase and its target sites frts form the amplification system which is responsible for the steady state plasmid copy number. Studies on the plasmid stability, plasmid copy number and endo 1,31,4 pglucanase production by free and alginate immobilised recombinant saccharomyces cerevisiae cells. Plasmids may be either high copy number plasmids or low copy number plasmids. Plasmid copy number and plasmid stability springerlink. Repe which controls the copy number of the minif plasmid. Plasmid copy number determines the gene dosage accessible for expression and many. Request pdf plasmid copy number and plasmid stability many expression systems in research and industry use plasmids as vectors for the production of.
We conclude that i instability and copy number vary greatly from plasmid to plasmid. On the other hand, if you get a poor yield from a high copy plasmid, then you know you have some troubleshooting to do, assuming your insert is not too large to begin with. Copy number variability of expression plasmids determined by cell. All of the model parameters for plasmid replication control can be obtained independently and no adjustable parameters are needed for the plasmid model. The relative plasmid copy number was obtained by comparing the intensity of the ethidium.
Guidance for industry considerations for plasmid dna vaccines for infectious disease indications additional copies of this guidance are available from the office of communication, training and. A mathematical model for prediction of plasmid copy number. It is convenient and desirable for the plasmid and chromosomal target fragments to be released by the same restriction enzyme. Increased plasmid copy number is essential for yersinia t3ss. Stability of the plasmid pkk223200 in escherichia coli jm105 was studied for both free and immobilized cells during continuous culture. To analyze an expression system the quantification of plasmid copy number is very helpful. Plasmid copy number determines the gene dosage accessible for expression and many plasmids lead generally to a high productivity. Estimation of copy number is made in comparison to known concentrations of a reference plasmid and also by comparison to quantitative dna markers. Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Plasmid copy number and plasmid stability researchgate.
As a model system we use inhibitordilution copy number control cnc of the bacterial plasmids cole1 and r1. It could be done by choosing high copy number plasmid as the expression vector. Guidance for industry considerations for plasmid dna vaccines. The promoter is not activated when it is desirable to maintain a copy number of one per haploid cell e. Although the plc replicon remained unaltered compared with its pbbr1 ancestor, the high copy number of plasmid phc comes at the cost of lower plasmid stability due to deletions of stability factors during construction of the puc replicon e. The procedure is based on southern hybridisation and measured the relative intensities of hybridisation of a probe to the single copy yeast chromosomal actin gene and to the multicopy plasmid pjg317. Materials and methods for controlling plasmid copy number. Evolution of plasmid stability under nonselective conditions provides an evolutionary explanation for the ubiquity of plasmids in nature. Plasmids carry a wide range of dispensable, transiently useful and often bizarre functions. The plasmid burden traditionally attributed to replication and maintenance of plasmid dna plays only a minor role in haploid yeast yet is much more significant in diploid strains. Characterization of a large, stable, highcopynumber. Isolation and characterization of novel mutations in the.
Direct and convenient measurement of plasmid stability in. Genetic control of cole1 plasmid stability that is. How to manipulate plasmid copy number bitesize bio. Bacterial plasmid stability summers 1985 bioessays.
Plasmids must regulate their copy number average number of plasmid copies per cell to ensure that they do not excessively burden the host or become lost during cell division. Plasmid copy number mutation in repa gene encoding repa replication initiator of cryptic plasmid phm1519 in corynebacterium glutamicum shuhei hashiro and hisashi yasueda institute for innovation, ajinomoto co. In direct contradiction with the classic random distribution model, we find no correlation between increased plasmid copy number and increased plasmid stability. An advantage of high copy number is the greater stability of the plasmid when random partitioning i. The chlamydia trachomatis plasmid is a virulence factor. Direct and convenient measurement of plasmid stability in lab and.
The same mutation also reduced the number of plasmid copies from to 5, as well as the plasmid stability. The higher the copy number is, the more likely the two daughter cells will contain the plasmid. Which dna purification technique involves purifying the sample using an ultracentrifuge. Copy number variability of expression plasmids determined by. The virulence of the plague bacterium, yersinia pestis, is encoded on a plasmid. Improved determination of plasmid copy number using. Jan 14, 2015 on the other hand, if you get a poor yield from a high copy plasmid, then you know you have some troubleshooting to do, assuming your insert is not too large to begin with. Copy number variability of expression plasmids determined. Owing to its high mitotic stability and copy number, components of the 2. Combinations of these modifications produce a range of copy numbers and of levels of recombinant expression. Jun 16, 2018 combinations of these modifications produce a range of copy numbers and of levels of recombinant expression. Determination of plasmid copy number and stability in. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. By using droplet digital pcr, plasmid copy number was found to be stable median, 5.
Jun, 2019 evolution of plasmid stability under nonselective conditions provides an evolutionary explanation for the ubiquity of plasmids in nature. Therefore, different methods for the determination of plasmid copy number are described. Plasmids are capable of transfer to other cells but not all plasmids are transferable. Guidance for industry considerations for plasmid dna. Dec 17, 2003 plasmid copy number determines the gene dosage accessible for expression and many plasmids lead generally to a high productivity. When a similar sequence in a cole1 derivative, pycw301, is mutated, the copy number of the plasmid also declines from 34 to 16 per cell. Schematic outline of plasmid stability model and its parameters.
However, the use of high copy number plasmid may lead to low stability, resulting in low cell productivity 14. Each plasmid has its independent replication system which controls the number of copies of the plasmid in a cell. Plasmid copy number and plasmid stability request pdf. What technique is used to increase plasmid copy number and purify plasmids. A recombinant yeast strain, saccharomyces cerevisiae dby746, containing the plasmid pjg317, was grown in a variety of. This mutation may be responsible for the increase in plasmid copy number. Methods and constructions for controlling the copy number and for maintaining the stability of a plasmid in yeast cells involving inserting into the plasmid a centromere sequence under the transcriptional control of a regulatable promotor. Plasmid stability in immobilized and free recombinant. Studies on the plasmid stability, plasmid copy number and. Multimerization of high copy number plasmids causes instability. Realtime pcr assays, using taqman chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcb gene in the infectious, elementary bodies ebs of c.
Here, we present a simple theoretical model to study plasmid stability, based on one input parameter which is the copy number of plasmids present in a host cell. Riboswitch signal amplification by controlling plasmid copy. Copy number and the stability of 2micron circlebased. The selection marker can also significantly change plasmid copy number.
A plasmid partition system is a mechanism that ensures the stable inheritance of plasmids during bacterial cell division. A desktop resource 1st edition 2 p a g e plasmids 101. Plasmid stability analysis based on a new theoretical model. A 75 kbp cryptic plasmid is found in almost all isolates of chlamydia trachomatis. Plasmid copy number and stability determination in. Studies on the plasmid stability, plasmid copy number and endo1, 31, 4 bglucanase production by free and alginate immobilised recombinant saccharomyces cerevisiae cells. Plasmid copy number average number of plasmids per bacterial cell was estimated using quantitative pcr on crude whole dna prepared from a midlog phase culture od 600 0. Larger plasmids are present in smaller numbers 12 and small plasmids may be found in high copy numbers 40. Because bla and dxs are single copy genes of pbr322 and lichromosomal dna, respectively, the ratio of bla to dxs is equal to the plas mid copy number of pbr322. Increased plasmid copy number is essential for yersinia. Plasmid stability 24 exists when, at cell division, each daughter cell receives at least one copy of the plasmid. The resulting variety of genetic ori modifications that we generated produced a range of plasmid copy numbers that allowed us to address whether plasmid copy number is a key determinant of plasmid stability in our system fig.