Plasmid-free Cells Research Articles

Flow cytometry as a useful tool for process development: rapid evaluation of expression systems

Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP) 260GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7- lac promoter and the P BAD promoter. The expression system based on the P BAD promoter was found to be superior to the T7-based system.

Origin pairing ('handcuffing') as a mode of negative control of P1 plasmid copy number.

In one family of bacterial plasmids, multiple initiator binding sites, called iterons, are used for initiation of plasmid replication as well as for the control of plasmid copy number. Iterons can also pair in vitro via the bound initiators. This pairing, called handcuffing, has been suggested to cause steric hindrance to initiation and thereby control the copy number. To test this hypothesis, we have compared copy numbers of isogenic miniP1 plasmid monomer and dimer. The dimer copy number was only one-quarter that of the monomer, suggesting that the higher local concentration of origins in the dimer facilitated their pairing. Physical evidence consistent with iteron-mediated pairing of origins preferentially in the dimer was obtained in vivo. Thus, origin handcuffing can be a mechanism to control P1 plasmid replication.

Forced periodic operations of continuous recombinant cell cultures subject to antibiotic selection pressure

Sustained production of recombinant proteins encoded by extrachromosomal plasmid DNA over long duration is not possible due to reversion of recombinant cells to unproductive plasmid-free cells. Application of antibiotic selection pressure, the most effective measure to overcome this problem, at a constant rate is prohibitively expensive. Nonlinearities in kinetics of recombinant bioprocesses provide an opportunity to possibly improve the time-average performance of these by periodic forcing of feed conditions. This possibility is investigated in this article using the generalized π-criterion for continuous recombinant cell cultures subject to periodic variations in one or more inputs (feed parameters), viz., dilution rate and feed concentrations of antibiotic and limiting substrate. The two types of antibiotic action considered are: (i) death of plasmid-free cells and (ii) repression of growth of plasmid-free cells. Pertinent analytical observations, which are applicable to continuous processes in general, are made about operations involving periodic forcing of multiple inputs. Where appropriate, analytical expressions are obtained for optimum amplitude ratios, optimum phase differences, and the corresponding maximum improvement in the process performance. The frequency intervals where an improvement in culture performance is guaranteed are identified via numerical illustrations for the two antibiotic types. These illustrations demonstrate that an increase in the number of feed parameters perturbed leads to broadening of the regions in the multidimensional parameter space where forced periodic operations are superior to steady-state operation. The π-criterion is also employed to investigate if forced periodic operation may enable retention of recombinant cells under conditions where such retention is not possible in a steady-state operation.

PRODUCTION OF PCB01, A PLASMID FOR DNA IMMUNIZATION AGAINST THE ADHESIN OF ESCHERICHIA COLI K88AB

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.

Postsegregational killing mediated by the P1 phage "addiction module" phd-doc requires the Escherichia coli programmed cell death system mazEF.

"Addiction modules" consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin. The extrachromosomal addiction module phd-doc, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). The Escherichia coli chromosomal addiction module analogue, mazEF, is responsible for the induction of programmed cell death. Here we show that the postsegregational killing mediated by the P1 phd-doc module depends on the presence of the E. coli mazEF system. In addition, we demonstrate that under conditions of postsegregational killing, mediated by phd-doc, protein synthesis of E. coli is inhibited. Based on our findings, we suggest the existence of a coupling between the phd-doc and mazEF systems.

Inclusion bodies as reversible protein reservoirs

Inclusion bodies (IBs) are major protein aggregates that arise owing to a deficiency of the cell's protein folding machinery. This often occurs in recombinant bacteria where plasmid-encoded genes are being highly expressed. In this context, research into the prevention of IB formation or the re-forming of correctly folded protein from IBs is of great importance to the biotechnology industry. Understanding IBs is equally important for research into amyloid diseases (such as Alzheimer's disease), which are caused by misfolding of proteins, resulting in their deposition in the cells as fibrils. Surprisingly, given the significance of IBs, they have generally been poorly investigated. However, a recent paper by Carrio et al. 1xCarrio, M.M et al. : 7–11See all References1 is an exception. They have shown that IB production in vivo is, in fact, caused by an imbalance between protein aggregation and solubility.The authors used two plasmid-encoded proteins expressed in Escherichia coli to study IB production. One was a β-galactosidase fused to a viral capsid protein, and the other was phage 22 tail-spike protein (TSP). In both systems, they calculated the number of IBs formed and their volume, the amount of soluble versus insoluble protein, and the activity of the protein as an indicator of proper folding. These measurements were taken at hourly intervals before and after the addition of chloramphenicol, which arrests protein synthesis. In the β-galactosidase system, using SDS-PAGE and densitometry against known standards, they showed that the amount of insoluble material in the cell cultures sharply decreased upon addition of chloramphenicol, with a concomitant increase in the amount of soluble material. A combination of microscopy and digital imaging also showed a reduction in the number and volume of IBs in the cells. Correct re-folding of the protein was confirmed by an increase in the β-galactosidase activity in the cultures following termination of protein synthesis. The same trend was found with the expression of TSP. Carrio et al. also demonstrated in vitro re-folding of IB proteins by incubating purified IBs from the two strains with cell extracts from plasmid-free BL26 cells, with the result being a recovery of enzymatic activity and soluble peptide in both systems.The authors concluded that IBs are the result of an imbalance in the equilibrium between protein precipitation and re-folding and solubility in the cell. By switching off protein production, the protein folding machinery can catch up, thus redressing the balance. The authors therefore see IBs not as a dead-end product caused by inefficiency of the bacterial protein folding machinery, but rather as a reversible reservoir of protein awaiting correct folding when conditions permit. The significance of the result is already apparent: IB solubility in vitro could provide the answer to the problem of IB formation in biotechnology and, from a clinical viewpoint, it raises possibilities for new therapeutic approaches to amyloid diseases.

Stable Maintenance of Plasmid in Continuous Culture of Yeast under Non-Selective Conditions.

A recombinant yeast plasmid containing the gene for beta-galactosidase was tested for stability in a host auxotrophic for leucine. Plasmid loss was studied at different dilution rates in continuous culture under selective as well as non-selective conditions. It was observed that the instability of the culture was higher at low dilution rates in selective medium, while the pattern was reversed when complex non-selective medium was used, with plasmid-containing cells competing effectively with plasmid-free cells at low dilution rates. This was attributed to a low residual yeast extract concentration in the medium at low dilution rates. Since yeast extract was the sole source of leucine, this limited the growth of plasmid-free cells, which were auxotrophic for leucine. Growth rate studies also indicated a competitive advantage of the plasmid-containing cells over the plasmid-free cells at low yeast extract concentrations in semi-defined medium. Using the above data, a modified continuous culture was run using non-selective medium at a low dilution rate of 0.05 h(-1). This resulted in stable coexistence of plasmid-containing and plasmid-free cells and hence sustained expression of beta-galactosidase at approximately 330 OD420l(-1) h(-1) throughout the period of cultivation (134 h).

Stable maintenance of plasmid in continuous culture of yeast under non-selective conditions

A recombinant yeast plasmid containing the gene for β-galactosidase was tested for stability in a host auxotrophic for leucine. Plasmid loss was studied at different dilution rates in continuous culture under selective as well as non-selective conditions. It was observed that the instability of the culture was higher at low dilution rates in selective medium, while the pattern was reversed when complex non-selective medium was used, with plasmid-containing cells competing effectively with plasmid-free cells at low dilution rates. This was attributed to a low residual yeast extract concentration in the medium at low dilution rates. Since yeast extract was the sole source of leucine, this limited the growth of plasmid-free cells, which were auxotrophic for leucine. Growth rate studies also indicated a competitive advantage of the plasmid-containing cells over the plasmid-free cells at low yeast extract concentrations in semi-defined medium. Using the above data, a modified continuous culture was run using non-selective medium at a low dilution rate of 0.05 h −1. This resulted in stable coexistence of plasmid-containing and plasmid-free cells and hence sustained expression of β-galactosidase at ∼330 OD 420 l −1 h −1 throughout the period of cultivation (134 h).

Plasmid maintenance and recombinant cell fitness explored in bacterial colonies

During growth of recombinant bacteria, irregular plasmid partitioning generates non-productive, plasmid-free cells whose proportion usually increases in the culture. For Escherichia coli producing engineered β-galactosidases, we have shown a coincidence between plasmid stability and the extension of white/blue areas within individual colonies on X-gal plates. In this context, a good correlation between plasmid permanence in colonies and parameters accurately describing the dynamics of plasmid-free cell population in liquid cultures has been observed. Moreover, the impact of lacZ gene engineering and the metabolic burden imposed by the encoded proteins has been evaluated through plasmid stability by simple image analysis, revealing an enhanced plasmid loss rate as the cells enter into the stationary phase that is modulated by the expression of particular recombinant genes.

Proteome analysis of a temperature-inducible recombinant Escherichia coli for poly-β-hydroxybutyrate production

A recombinant Escherichia coli strain harboring the λ p R- p L promoter and heterologous poly-β-hydroxybutyrate (PHB) biosynthesis genes was shown to accumulate PHB when the incubation temperature was changed from 34°C to temperatures higher than 37°C. In the present research, total gene expression patterns of the recombinant E. coli before and after induction were investigated by two-dimensional gel electrophoresis. Proteins encoded by serS, sucC, trpA, and alaS were found to be expressed before induction of phb genes at a culture temperature of 34°C. On the other hand, proteins encoded by metG, rplI, and carA were found to be expressed after induction achieved by increasing the temperature to 40°C. In the case of plasmid-free cells, all the selected genes have been shown to be expressed except metG, and ibpA and ibpB among the heat-shock proteins. The heat-shock proteins were found to be upregulated upon induction of phb genes, which may be due to the stress caused by the accumulation of PHB granules as well as by the temperature upshift. The changes in the expression of some of the metabolic pathway-related proteins before and after induction were interpreted in relation to the consumption of NADPH and acetyl-CoA for PHB synthesis.

Degradation of Mineral Oil with a Strain of Acinetobacter calcoaceticus

The Acinetobacter calcoaceticusstrain TM-31 has been isolated from a microbial assemblage of a pilot plant that purifies waste water polluted with mineral oil. This strain is capable of efficient degradation of components of mineral oil (alkanes, isoalkanes, and alkyl residues of the naphthene and arene fraction). The strain bears stably inherited plasmids of sizes 120, 9, and 8 kb, which can be transferred into plasmid-free cells of the parental strain and into bacteria of the genusPseudomonasand ensure the degradation of hexadecane and mineral oil.

Proteome Analysis of a Temperature-Inducible Recombinant Escherichia coli for Poly-.BETA.-Hydroxybutyrate Production.

A recombinant Escherichia coli strain harboring the lambdap(R)-p(L) promoter and heterologous poly-beta-hydroxybutyrate (PHB) biosynthesis genes was shown to accumulate PHB when the incubation temperature was changed from 34 degrees C to temperatures higher than 37 degrees C. In the present research, total gene expression patterns of the recombinant E. coli before and after induction were investigated by two-dimensional gel electrophoresis. Proteins encoded by serS, sucC, trpA, and alaS were found to be expressed before induction of phb genes at a culture temperature of 34 degrees C. On the other hand, proteins encoded by metG, rplI, and carA were found to be expressed after induction achieved by increasing the temperature to 40 degrees C. In the case of plasmid-free cells, all the selected genes have been shown to be expressed except metG, and ibpA and ibpB among the heat-shock proteins. The heat-shock proteins were found to be upregulated upon induction of phb genes, which may be due to the stress caused by the accumulation of PHB granules as well as by the temperature upshift. The changes in the expression of some of the metabolic pathway-related proteins before and after induction were interpreted in relation to the consumption of NADPH and acetyl-CoA for PHB synthesis.

Two-stage cultivation of recombinant Saccharomyces cerevisiae to enhance plasmid stability under non-selective conditions: experimental study and modeling.

A leucine auxotroph strain of Saccharomyces cerevisiae was used to study plasmid stability and expression using a recombinant plasmid, which contained a foreign gene for firefly luciferase (luc). This recombinant yeast was tested in a series of continuous cultures in semi-defined media with varying concentrations of yeast extract in order to study its effect on stability. While the biomass concentration and luciferase activity increased with increasing concentrations of yeast extract, the plasmid stability declined. An analysis of the growth rates showed that the recombinants enjoyed a growth rate advantage over the plasmid-free cells at critically low yeast extract concentrations, possibly due to leucine starvation in the media. A two-stage cultivation strategy was designed in order to create a yeast extract limited environment so that plasmid-free cells could not grow and overtake the recombinant cells. The cells were cultivated in selective media in the first stage, and then transferred continuously to the second stage where the media was enriched by feeding yeast extract. The feed rate was kept low in order to ensure yeast extract and hence leucine starvation, thereby selecting against the plasmid-free cells. This strategy resulted in a stable existence of recombinant cells, which stabilized around 60% at steady state during the tested period of cultivation. The complex nitrogen feed helped in increasing the cell density and volumetric activity by approximately 9 and 18-fold respectively with respect to that achieved in minimal medium. The experimental data was used to formulate a mathematical model to predict cell growth and plasmid stability in two-stage cultivation, which correctly explained the experimental data.

Cell-associated pheromone peptide (cCF10) production and pheromone inhibition in Enterococcus faecalis.

In Enterococcus faecalis, the peptide cCF10 acts as a pheromone, inducing transfer of the conjugative plasmid pCF10 from plasmid-containing donor cells to plasmid-free recipient cells. In these studies, it was found that a substantial amount of cCF10 associates with the envelope of the producing cell. Pheromone activity was detected in both wall and membrane fractions, with the highest activity associated with the wall. Experiments examining the effects of protease inhibitor treatments either prior to or following cell fractionation suggested the presence of a cell envelope-associated pro-cCF10 that can be processed to mature cCF10 by a maturase or protease. A pCF10-encoded membrane protein, PrgY, was shown to prevent self-induction of donor cells by reducing the level of pheromone activity in the cell wall fraction.

The antisense RNA of the par locus of pAD1 regulates the expression of a 33-amino-acid toxic peptide by an unusual mechanism.

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame of 33 codons designated fst. The results presented here demonstrate that the peptide encoded by fst is the par toxin. The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the fst-encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem-loop structure at the 5' end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fold increase in production of the fst-encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of fst in plasmid-free cells.

Influence of controlled glucose oscillations on a fed-batch process of recombinant Escherichia coli

The influence of glucose oscillations on cell growth and product formation of a recombinant Escherichia coli culture producing a heterologous α-glucosidase was studied in fed-batch cultures in a laboratory bioreactor. Glucose oscillations were created by an on/off-feeding mode in either fast cycles (1 min) or slow cycles (4 min) and compared to a process with constant glucose addition. The study indicates that glucose oscillations influence the product stability and the overgrowth of plasmid-free cells if such cultures are not performed under continuous pressure for selection of plasmid-containing cells. Although the glucose uptake capacity decreased after induction of the recombinant α-glucosidase in all cultures performed, the up-growth of plasmid-free cells during the production phase was strongly inhibited by fast oscillations. In contrast, plasmid-free cells grew up when constant feeding or slow cycles were applied. Our data suggest that the various feed protocols effect the specific carbon dioxide formation rate differently, with the highest production of carbon dioxide in the cultivations with fast cycles. In connection to product formation the initial α-glucosidase accumulation was the same in all cultures, but the stability of the product was significantly lower in the cultivation with slow cycles. Our results from laboratory experiments are discussed in relation to the mixing situation in large-scale bioreactors.

Cell size differences in plasmid-containing and plasmid-free cells of Bacillus subtilis during batch and cyclic batch culture

Differences in cell sizes were observed in recombinant Bacillus subtilis studied in cyclic batch culture. It was observed that under this mode of operation both plasmid-containing and plasmid-free cells were present in the culture broth for an extended period of time when compared to batch culture. In cyclic batch culture, plasmid-containing cells decreased from 100% to 0% over a period of 75 h, compared with batch culture where plasmid-containing cells decreased to 0 over a period of 20 h. Plasmid copy number was found to be similar in cyclic batch culture to that found in batch culture. Copy number increased from 90 copies up to a maximum of 190 copies in cyclic batch culture after the drop in plasmid-containing cells below 40%. In batch culture, copy number ranged from 90 to 140. Cell size distributions by using image analysis and particle-size equipment indicated the existence of two populations in cyclic batch culture. Average cell size decreased with decreasing percentage of plasmid-containing cells, whereas the average plasmid-containing cell size increased with increasing plasmid copy number.

Metastable structures and refolding kinetics in hok mRNA of plasmid R1.

Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5' end. Here, experimental evidence is presented for the existence of metastable structures in the 5' leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5' ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription.

Identification and characterization of a determinant (eep) on the Enterococcus faecalis chromosome that is involved in production of the peptide sex pheromone cAD1.

Plasmid-free strains of Enterococcus faecalis secrete a peptide sex pheromone, cAD1, which specifically induces a mating response by donors carrying the hemolysin plasmid pAD1 or related elements. A determinant on the E. faecalis OG1X chromosome has been found to encode a 46.5-kDa protein that plays an important role in the production of the extracellular cAD1. Wild-type E. faecalis OG1X cells harboring a plasmid chimera carrying the determinant exhibited an eightfold enhanced production of cAD1, and plasmid-free cells carrying a mutated chromosomal determinant secreted undetectable or very low amounts of the pheromone. The production of other pheromones such as cPD1, cOB1, and cCF10 was also influenced, although there was no effect on the pheromone cAM373. The determinant, designated eep (for enhanced expression of pheromone), did not include the sequence of the pheromone. Its deduced product (Eep) contains apparent membrane-spanning sequences; conceivably it is involved in processing a pheromone precursor structure or in some way regulates expression or secretion.

Bistability of Stationary States During Competition between Plasmid-Bearing and Plasmid-Free Cells under Selection Pressure

Feasibility and stability analyses have been carried out for the stationary states of a chemostat with recombinant microorganisms. In the absence of selection pressure, only a null state is possible, as expected. With selection pressure exerted by an antibiotic, either of two states may exist, according to the combination of dilution rate and inlet concentration. This is true for yeasts (S. cerevisiae) as well as bacteria (E. coli). However, too strong an antibiotic concentration kills all plasmid-free cells, thereby disallowing a non-trivial stationary state. Accounting for variation of the plasmid loss probability with the specific growth rate increases the stability regions. This is also possible by allowing a higher rate of segregational plasmid loss, implying that genetic stability may have to be balanced against reactor stability.