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This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed in a one-week period includes the protocols for plasmid pVAX1-LacZ production in Escherichia coli DH5α cells and subsequent purification of supercoiled pVAX1-LacZ. Firstly, the students use a growth medium that favors the replication of the plasmid resulting in a higher plasmid production during exponential growth. Afterwards, alkaline lysis is done to disrupt the bacterial cells and recover pVAX1-LacZ plasmid. Lastly, they perform the purification of pVAX1-LacZ supercoiled isoform by L-histidine chromatography, followed by agarose gel electrophoresis to characterize the separation of supercoiled isoform from contaminants. The proposed experiment provides an opportunity for students to acquire these skills that are routinely used in biochemistry and biotechnology laboratories. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):638-643, 2019.

We investigated the cultivation of bacterial, mammalian and insect cells in an orbitally-shaken 250-mL disposable tube that incorporates a helical track (HT) on its inside wall. The mass transfer coefficient of oxygen (kLa) was 200%–400% higher in the HT tube than in a shake flask. Bacterial growth and plasmid production were 30% higher in the HT tube than in a 1-L Erlenmeyer flask. Mammalian cell cultures achieved a 25% higher cell density in the HT tube as compared to a 250-mL square-shaped bottle while insect cells grew as well in HT tubes as in 250-mL shake flasks. Because of their performance, disposability, and compact size, we conclude that 250-mL HT tubes are a useful alternative to other shaken containers for the cultivation of bacterial, mammalian and insect cells.

Growth characteristics and plasmid yields of Escherichia coli JM109 transformed with pCB01, a plasmid that encodes genes for the fimbrial adhesin of E. coli K88ab and for ampicillin resistance, grown in two culture media in agitated flasks and in fermentor, are reported. The rate of plasmid loss during growth was estimated by the differential counts in media with and without ampicillin. Plasmid yields of cultures grown in flasks varied from 0.9 to 67 µg/ml of medium, while those grown in fermentor attained 62 µg/ml of medium after 8 hours of culture. Plasmid bearing cells were outgrown by plasmid free cells in proportions varying from 5 to 1.2 non-transformed for each transformed cell during growth. Generation times of total population and plasmid bearing cells were 33 and 61 minutes, and 36 and 121 minutes, for fermentor and flask grown cultures, respectively. The same culture grown in fermentor and in flasks produced 62 and 33 µg of plasmid DNA per ml of medium, respectively. Bacterial concentrations and plasmid yields were higher in BHI than in LB medium. Yields of plasmid DNA obtained from the same batch were 1,7 times higher with cesium chloride-ethidium bromide gradients than with commercial columns. Comunicam-se algumas características de cultivo e a produção de plasmídios por Escherichia coli JM109 transformada com pCB01, um plasmídio que codifica os genes da adesina fimbrial de E. coli K88ab e de resistência à ampicilina. A cepa foi cultivada em Infuso de Cérebro e Coração (BHI) e em caldo de Luria (LB), em Erlenmeyers agitados e em fermentador de bancada. A taxa de perda de plasmídios foi estimada pela diferença de contagens em meio com e sem ampicilina. Os rendimentos de plasmídios das culturas em Erlenmeyer variaram de 0,9 a 67 µg/ml de meio, entanto as cultivadas em fermentador alcançaram 62 µg/ml após 8 horas de cultivo. Células sem plasmídio superaram às transformadas de 5 a 1,2 vezes durante o cultivo. Os tempos de geração da população total e das células transformadas foram 33 e 61 minutos no fermentador, e 36 e 121 minutos em Erlenmeyers, respectivamente. O mesmo inoculo produziu 62 e 33 µg de plasmídio por ml de meio no fermentador e em Erlenmeyers, respectivamente. As concentrações de bactérias e o rendimento de plasmídios foram superiores em BHI que em LB. O rendimento de plasmídios obtidos do mesmo cultivo foram 1,7 vezes maiores quando a purificação foi feita pelo método de ultracentrifugação em gradientes de césio-brometo de etídio do que com colunas comerciais.

Growth characteristics and plasmid yields of Escherichia coli JM109 transformed with pCB01, a plasmid that encodes genes for the fimbrial adhesin of E. coli K88ab and for ampicillin resistance, grown in two culture media in agitated flasks and in fermentor, are reported. The rate of plasmid loss during growth was estimated by the differential counts in media with and without ampicillin. Plasmid yields of cultures grown in flasks varied from 0.9 to 67 µg/ml of medium, while those grown in fermentor attained 62 µg/ml of medium after 8 hours of culture. Plasmid bearing cells were outgrown by plasmid free cells in proportions varying from 5 to 1.2 non-transformed for each transformed cell during growth. Generation times of total population and plasmid bearing cells were 33 and 61 minutes, and 36 and 121 minutes, for fermentor and flask grown cultures, respectively. The same culture grown in fermentor and in flasks produced 62 and 33 µg of plasmid DNA per ml of medium, respectively. Bacterial concentrations and plasmid yields were higher in BHI than in LB medium. Yields of plasmid DNA obtained from the same batch were 1,7 times higher with cesium chloride-ethidium bromide gradients than with commercial columns.Comunicam-se algumas características de cultivo e a produção de plasmídios por Escherichia coli JM109 transformada com pCB01, um plasmídio que codifica os genes da adesina fimbrial de E. coli K88ab e de resistência à ampicilina. A cepa foi cultivada em Infuso de Cérebro e Coração (BHI) e em caldo de Luria (LB), em Erlenmeyers agitados e em fermentador de bancada. A taxa de perda de plasmídios foi estimada pela diferença de contagens em meio com e sem ampicilina. Os rendimentos de plasmídios das culturas em Erlenmeyer variaram de 0,9 a 67 µg/ml de meio, entanto as cultivadas em fermentador alcançaram 62 µg/ml após 8 horas de cultivo. Células sem plasmídio superaram às transformadas de 5 a 1,2 vezes durante o cultivo. Os tempos de geração da população total e das células transformadas foram 33 e 61 minutos no fermentador, e 36 e 121 minutos em Erlenmeyers, respectivamente. O mesmo inoculo produziu 62 e 33 µg de plasmídio por ml de meio no fermentador e em Erlenmeyers, respectivamente. As concentrações de bactérias e o rendimento de plasmídios foram superiores em BHI que em LB. O rendimento de plasmídios obtidos do mesmo cultivo foram 1,7 vezes maiores quando a purificação foi feita pelo método de ultracentrifugação em gradientes de césio-brometo de etídio do que com colunas comerciais.

We describe a generic plasmid purification process for producing DNA for larger-scale transient transfection. Data on plasmid quality with regard to residual protein, endotoxin content and presence of different plasmid forms is given. The effects of contaminants and plasmid forms on expression levels of TNFRp55 and SEAP are discussed. Transient transfection of serum-free suspension grown mammalian cells represents a suitable approach to provide research quantities of proteins (50–100 mg) within1–2 weeks.

The standard procedure in the lab for plasmid isolation usually involves a 2-mL, 16 h over-night cultivation in 15-mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high-throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5-mL microcentrifuge tube with only 100 μL cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5–4 μg mL(–1), a 6- to 10-fold increase in plasmid concentration (from 10 up to 25 μg mL(–1) cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase®). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed a simple protocol with a significantly decreased procedure time, enabling simultaneous handling of more samples, while a consistent quality and a higher final pDNA concentration are ensured.

Monitoring plasmid production systems is a lab intensive task. This article proposes a methodology based on FTIR spectroscopy and the use of chemometrics for the high-throughput analysis of the plasmid bioproduction process in E. coli. For this study, five batch cultures with different initial medium compositions are designed to represent different biomass and plasmid production behavior, with the maximum plasmid and biomass concentrations varying from 11 to 95 mg L−1 and 6.8 to 12.8 g L−1, respectively, and the plasmid production per biomass varying from 0.4 to 5.1 mg g−1. After a short sample processing consisting of centrifugation and dehydration, the FTIR spectra are recorded from the collected cellular biomass using microtiter plates with 96 wells. After spectral pre-processing, the predictive FTIR spectra models are derived by using partial least squares (PLS) regression with the wavenumber selection performed by a Monte-Carlo strategy. Results show that it is possible to improve the PLS models by selecting specific spectral ranges. For the plasmid model, the spectral regions between 590–1,130, 1,670–2,025, and 2,565–3,280 cm−1 are found to be highly relevant. Whereas for the biomass, the best wavenumber selections are between 900–1,200, 1,500–1,800, and 2,850–3,200 cm−1. The optimized PLS models show a high coefficient of determination of 0.91 and 0.89 for the plasmid and biomass concentration, respectively. Additional PLS models for the prediction of the carbon sources glucose and glycerol and the by-product acetic acid, based on metabolism-induced correlations between the nutrients and the cellular biomass are also established. Biotechnol. Bioeng. 2012;109: 2279–2285. © 2012 Wiley Periodicals, Inc.

Application of plasmid DNA as pharmaceutical to be used in gene therapy and vaccination has been investigated intensively in recent years. To be able to provide sufficient material that is in accordance with quality of pharmaceutical grade it is mandatory to gain comprehensive process knowledge which is even requested by regulatory agencies. Regarding plasmid DNA production the specific growth rate has been identified as one of the key parameters. The reduction of specific growth rate results in an increase of plasmid DNA formation. However, quantitative explanations that allow for efficient process development and design are still missing. The presented study proposes a model that clearly demonstrates the relationship between specific growth rate and plasmid formation due to identification of the specific plasmid production rate as relevant key parameter. In addition the model is proved to serve as a useful tool in process development and design.

A generalized regression neural network with external feedback was used to predict plasmid production in a fed-batch cultivation of recombinant Escherichia coli. The neu ral network was built out of the experimental data obtained on a few cultivations, of which the general strategy was based on an initial batch phase followed by an exponen tial feeding phase. The different cultivation conditions used resulted in significant differ ences in bacterial growth and plasmid production. The obtained model allows estimation of the experimental outputs (biomass, glucose, acetate and plasmid) based on the bioreactor starting conditions and the following on-line inputs: feeding rate, dissolved oxygen concentration and bioreactor stirring speed. Therefore, the proposed methodol ogy presents a quick, simple and reliable way to perform on-line feedback prediction of the dynamic behaviour of the complex plasmid production process, based on simple on-line input data obtained directly from the bioreactor control unit and with few cultiva tion experiments for neural network learning.

In 1990 a new approach for vaccination was invented involving injection of plasmid DNA in vivo, which elicits an immune response to the encoded protein. DNA vaccination can overcome most disadvantages of conventional vaccine strategies and has potential for vaccines of the future. However, today 15 years on, a commercial product still has not reached the market. One possible explanation could be the technique's failure to induce an efficient immune response in humans, but safety may also be a fundamental issue. This review focuses on the safety of the genetic elements of DNA vaccines and on the safety of the microbial host for the production of plasmid DNA. We also propose candidates for the vaccine's genetic elements and for its microbial production host that can heighten the vaccine's safety and facilitate its entry to the market.

Cost-effective production of commercial-scale batches of pharmaceutical-grade plasmid DNA remains a challenge for the maturing industry of plasmid-based biologics. A process is required that meets the quality standards for human pharmaceutical products and yet is economically suitable for use in veterinary medicine or as an in-process product for viral therapeutic production. Previously described scaleable cell lysis requires a substantial capital investment. Also, these techniques contaminate the process stream with unnecessary impurity levels. A simple, effective purification strategy requires elimination of all animal-derived enzymes, without adding costly column-based chromatography steps. The method described herein has produced multi-gram batches of pharmaceutical-grade plasmid at a fraction of the cost of previously described methods.

A generalized regression neural network with external feedback was used to predict plasmid production in a fed-batch cultivation of recombinant Escherichia coli. The neu ral network was built out of the experimental data obtained on a few cultivations, of which the general strategy was based on an initial batch phase followed by an exponen tial feeding phase. The different cultivation conditions used resulted in significant differ ences in bacterial growth and plasmid production. The obtained model allows estimation of the experimental outputs (biomass, glucose, acetate and plasmid) based on the bioreactor starting conditions and the following on-line inputs: feeding rate, dissolved oxygen concentration and bioreactor stirring speed. Therefore, the proposed methodol ogy presents a quick, simple and reliable way to perform on-line feedback prediction of the dynamic behaviour of the complex plasmid production process, based on simple on-line input data obtained directly from the bioreactor control unit and with few cultiva tion experiments for neural network learning.