A method for the stabilisation of recombinant plasmids in yeast.

The stability of a yeast plasmid can be improved using deliberately induced cyclic changes in the dissolved oxygen tension (DOT), during continuous culture in a non-selective, undefined medium. The resultant stability of the plasmid under DOT cycled conditions is strongly dependent on the growth rate of the culture, with complete stabilisation at lower growth rates. We propose a mechanism for the stabilisation and suggest that the method can be applied to other recombinant yeast strains.     

Kinetic study of instability of recombinant plasmid pPLc23trpAl in E. coli using two-stage continuous culture system

Derepression of the phage lambda p(L) promoter on recombinant plasmid pPLc 23-trpAl caused a rapid increase of plasmid free segregants in the population. In continuous culture, increased production of trpA protein follwing derepression was accompanied by a continuous deceleration of specific growth rate. In the repressed condition, plasmid loss per generation in continuous culture decreased as dilution rate increased from 0.06 to 1.08 h(-1). Over this range, the concentration of plasmid DNA within the cell decreased eightfold corresponding to a decrease in plasmid number from 74 to 32 molecules/cell. The use of a two-stage continuous culture system coupled with a temperature sensitive expression system allows a high trpA productivity from the derepressed plasmid for more than 48 h and also offers a possibility of minimizing the instability problem of high expression recombinants. Such a system also permits the critical study of the effects of fermentation and other regulatory parameters on expression under better controlled conditions than is possible in a batch culture or single-stage continous culture.     

Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye

A sensitive fluorescence-based method for monitoring plasmid DNA during production was investigated. This simple method of assaying for plasmid DNA allows rapid monitoring of plasmid yields from a recombinant Escherichia coli fed-batch fermentation. The assay has several advantages over traditional methods of plasmid DNA measurement. The fluorescent dye is highly specific and can measure total plasmid DNA concentration in about 5 min. The assay is sensitive over a wide range of plasmid concentrations of between 15 and 280 ng/mL, even in the presence of impurities that occur within alkaline lysate preparations. The technique can also be applied to monitoring fermentation and downstream purification steps.     

Expression of recombinant protein A from the lac promoter in Escherichia coli JM83 is not subject to catabolite repression when grown under specific conditions of continuous culture

Although widely used in experimental and industrial situations, genetically engineered plasmids containing the lac promoter from Escherichia coli are subject to catabolite repression when grown in glucose-containing media. Several methods of overcoming this problem have been investigated by studying the expression of the protein A gene from Staphylococcus aureus under the control of the Escherichia coli lac promoter. When glycerol is used as a sole carbon source, the plasmid is unstable and is rapidly lost from the culture. When the bacteria are grown in chemostats under glucose limitation, the plasmid is maintained, even at high dilution rates, and the expression of protein A is similar to that observed when glycerol was used. The balance between metabolic load and protein A expression seems to be maintained by reducing the gene dose to a tolerable level. Depending on the metabolic conditions prevailing in the culture, this is achieved, either by reducing the copy number of the plasmid or in extreme cases by removing the plasmid altogether.     

Construction of vector of Brevibacterium lactofermentum and study of its stability in continuous culture.

A 5.7-kb vector plasmid pBK2 was constructed by ligating the kanamycin resistance gene from Escherichia coli plasmid pACYC177 to an endogenous cryptic 4.4-kb plasmid of Brevibacterium lactofermentum ATCC 21086. The vector replicates efficiently and is stably maintained in the host and other coryneforms. However, the copy number varied from 50 to 10 per chromosome-equivalent under different culture conditions. Continuous culture studies showed instability when low dilution rates were used. Co-culture experiments were performed at various dilution rates to measure the growth rate ratio (alpha) of the plasmid-free cells to the plasmid-containing cells. It was observed that at low dilution rates the value of alpha was higher than that at high dilution rates. Thus, the instability of the plasmid can be attributed to the increase in alpha at low dilution rates. Modelling of instability using a random partitioning model of plasmid segregation and experimentally obtained values of alpha showed agreement with experimental data. This demonstrated that active partitioning is not the operative mechanism for plasmid segregation in this case.     

Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum.

A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation. The method can be scaled up, without difficulty, for large-scale plasmid preparation.     

Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum

A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation. The method can be scaled up, without difficulty, for large-scale plasmid preparation.     

The nucleotide sequence of a mitochondrial replicon from maize

The 1913-bp maize mitochondrial (mt) plasmid was isolated from a suspension culture of a Black Mexican Sweet maize strain, cloned into M13mp vectors, and sequenced by a unidirectional progressive deletion method. The 1.9-kb extrachromosomal double-stranded circular DNA plasmid was found to contain regions of sequence which in other systems are known to be part of origins of replication (ori). This plasmid could be used as a carrier for chimeric genes and a molecular probe for replication.     

Estimating the rate of plasmid transfer: an end-point method

We describe a method for determining the rate parameter of conjugative plasmid transfer that is based on single estimates of donor, recipient and transconjugant densities and the growth rate in exponential phase of the mating culture. The formula for estimating the plasmid transfer rate, gamma, was derived from a mathematical model describing cell growth and plasmid transfer in batch culture. Computer simulations were used to explore the sensitivity of this method to the realities of bacterial life, such as growth rate differences, plasmid segregation and transitory derepression of pilus synthesis. As predicted by the theory, mating experiments with the plasmid R1 in Escherichia coli K12 demonstrated that the estimate gamma is unaffected by cell density, donor:recipient ratio and mating time. Unlike previous techniques, our method allows us to investigate the effect of environmental factors on plasmid transfer rates when these factors also influence population growth rates. To illustrate this, we examined the effect of temperature on the rate of plasmid transfer.     

Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier

A simple method for plasmid minipreps in closed 1.5 mL microcentrifuge tubes using a cultivation medium with internal substrate delivery (EnBase^®) in combination with a two-phase perfluorodecalin (PFD) system supplying additional oxygen to the E. coli culture is described. The procedure can simply be performed on a thermoshaker using only 50 μL cultivation volume. Twenty and twenty-five percent higher cell densities and plasmid concentration, respectively, were obtained with the additional oxygen delivery system when compared to cultures without PFD. Compared to standard 2 mL LB cultures ninefold higher cell densities and eightfold higher plasmid concentrations were achieved for the smaller culture volume. The μL-scale cultures can be directly utilized in further plasmid purification without any centrifugation step or the subsequent removal of the supernatant. This simplifies the routine procedure considerably. Furthermore, the new method is very robust considering the time of cultivation. Highest plasmid concentrations were already obtained after only 6 h of cultivation, but the plasmid concentration remained high (87 % of the maximum) even until 8 h of cultivation. Aside from the advantage of this method for the daily routine, we believe that it could also be applied to automated high-throughput processes.     

Purification of plasmid DNA by tangential flow filtration

A simple, scalable method for purification of plasmid DNA is described. The method includes modification of the classical alkaline-lysis-based plasmid extraction method by extending the solubilization step from less than 30 min to 24 h. The extraction is followed by the novel use of tangential flow filtration (TFF) for purification of the remaining contaminants. The method does not include the use of any organic solvents, RNase, high-speed centrifugation, or column chromatography steps. The method typically yields 15 to 20 mg of plasmid DNA per liter of bacterial culture and results in removal of >99% of RNA and >95% of the protein that remains after the modified alkaline lysis procedure. The procedure has been demonstrated to be effective in the isolation of seven different plasmids. Plasmids isolated using this method had comparable transfection capability relative to plasmid isolated using a classical, cesium chloride gradient-based method.     

Escherichia coli plasmid production in fermenter

Escherichia coli harboring a recombinant plasmid was grown in a fermenter to study the effects of selected process parameters on the growth of the microbe and on plasmid amplification with chloramphenicol treatment. Eighteen fermentations were carried out according to a statistical experimental design in which the fermentation temperature, pH, and turbidity of culture at the onset of plasmid amplification were the selected independent process variables. Static regression models describing the process were derived from the experimental results. It turned out that recombinant plasmid copy numbers could be influenced by controlling fermentation temperature and pH. The maximal copy number during bacterial growth phase and the optimal plasmid production were found to require fermentation conditions different from those needed for optimal bacterial growth and cell division. The conditions also differed significantly from those routinely used in research laboratories for plasmid preparation. The chloramphenicol treatment increased the plasmid copy number compared with chromosome numbers up to fivefold. Some of the data suggest that under certain conditions the number of chromosome molecules in E. coli cells may rise during the plasmid amplification stage. Statistical experimental design, a nucleic acid sandwich hybridization technique for plasmid quantification, and regression models proved to be useful in this study.     

Microplate bioassay for nisin in foods,based on nisin-induced green fluorescent protein fluorescence

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 microg of nisin in cheese, and 1 microg of nisin per ml in salad dressings.     

Plasmid contents of lactic acid bacteria isolated from different types of sour doughs

The plasmid contents of 13 lactic acid bacteria isolated from different types of sour doughs were examined and compared with the plasmid contents of 11 culture collection strains and one commercial pure starter culture for sour doughs. In addition, plasmid analysis was used as a tool to study the stability of a starter culture during sour dough fermentation in a bakery.The tested strains varied in plasmid content from no plasmid up to six plasmids, with molecular weights from 1.5 to 43 MDal. In most cases, the wild-type strains contained a higher number of plasmids than the culture collection strains. The ability of the strains to ferment different carbohydrates was also investigated, but no obvious correlations between the fermentation patterns and the plasmid patterns could be observed. During the fermentation of the bakery sour dough, strains other than the inoculated starter culture gradually became dominant in the microflora. These new strains contained 1–3 plasmids, contrary to the plasmidless starter culture, and they also fermented more carbohydrates than the starter culture.     

Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P in continuous culture

Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P, a strain capable of mineralizing 3- and 4-chlorobenzoate and 4-methylbenzoate, was investigated in continuous culture. The hybrid cosmid pFRC20P enables the strain to mineralize 4-methylbenzoate. Rapid plasmid loss was observed under nonselective conditions using 3-chlorobenzoate as the substrate. Plasmid stability decreased with increasing dilution rate. Despite the growth advantage of the generated plasmid free cells a total depletion of plasmid bearing cells was not observed. After approximately 50 generations the fraction of plasmid bearing cells reached a constant level of 10%, which was stably maintained during the next 25 generations. Cells from this stage were used to inoculate a new culture that resulted in a stable level of 50% plasmid bearing cells. By a temporary substrate change to selective conditions (4-methylbenzoate), this level could be further increased to 70%. Literature models on plasmid stability could not be applied to describe the experimental data. Therefore, a new but unstructured model was developed to describe the experimental results. The model is based on the existence of three subpopulations: a plasmid free one, an original plasmid bearing one with a growth disadvantage compared to plasmid free cells, and a second plasmid bearing subpopulation with increased stability that is generated from the original one and has a growth rate comparable to the plasmid free cells.     

Production and Properties of α-l-Arabinofuranosidase from Various Strains of Corticium rolfsii

Human erythropoietin (Epo) cDNA was engineered for expression in cultured tobacco cells (Nicotiana tabacum L. cv. BY2). Two plasmid DNAs were constructed: pCEP, which contained Epo cDNA under control of the cauliflower mosaic virus-derived 35S RNA promoter and terminator, and pNSEP, which contained signal sequence-deleted Epo cDNA under control of the 35S RNA promoter and terminator. By using the electroporation method, each of these plasmid DNAs was transferred into the protoplasts of BY2 cells together with a plasmid, pNR35, which conferred G418-resistance on the cells. Four G418-resistant clones were obtained from protoplasts transfected with pNSEP and pNR35, and only one of them, named 11N, survived in suspension culture. Integration of pNSEP DNA into the genome of 11N cells was confirmed by Southern blot and PCR analyses. Production of Epo mRNA was shown by Northern blot analysis. Epo protein was shown to be expressed in 11N cells by colorimetric enzyme immunoassay. The productivity of Epo in the 11N cells (1 pg/g of wet cells) was very low.     

Retrotransfer of DNA in the rhizosphere

Retrotransfer of DNA refers to the phenomenon by which a plasmid travels from a host strain to a recipient one and returns to the original host, bringing with it DNA from the recipient. The resultant host strain with DNA from the recipient is called a retrotransconjugant. The retrotransfer phenomenon mediated by the TOL plasmid pWW0 and other plasmids has been documented on plates under optimal laboratory culture conditions, but never under natural conditions. In this work, we show that retrotransfer mediated by the IncP9 TOL pWW0 plasmid occurs in the rhizosphere, a niche in which the continuous supply of nutrients via root exudates allows cells to reach a high density. This suggests that this unusual sexual fertilization may be of great importance in lateral gene transfer. We also show that retrotransfer of DNA seems to require co-integration of the plasmid and the host chromosome and subsequent resolution, because a TOL plasmid with a mutation in the tnpR gene, encoding the resolvase of the Tn 4653 of the TOL plasmid, was self-transferred between Pseudomonas strains, but unable to mobilize chromosome.     

Stability of a lac operon in Zymomonas mobilis in batch and continuous culture

ZM6100(RP1::Tn951), a strain of Zymomonas mobilis containing the lactose transposon Tn951 on the broad host range plasmid RP1, progressively lost all plasmid markers in batch culture under non-selective conditions. After 120 generations less than 0.1% of the population retained the plasmid markers. ZM6306, derived from ZM6100(RP1::Tn951) by prolonged tetracycline selection, showed 100% stability for all plasmid markers when grown without selection pressure in both batch and continuous culture. In continuous culture, the synthesis of β-galactosidase was induced by the addition of lactose, and low levels of galactose were detected together with a small increase in ethanol concentration.     

Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges.     

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.