Study of population dynamic for a recombinant bacterium during continuous cultures: application of data filtering and smoothing

A numerical method to process experimental data concerning plasmid stability of a recombinant bacteria during continuous cultures with nonselective media is proposed here. This method differs from previous ones in that it uses the derivatve form of the state equation of the Imanaka-Aiba model for recombinant cultures. The methodology proposed here allows one to estimate values for the two model parameters without forcing them to be constant. Until now, this could not be done using classical analytical techniques because these parameters have been considered invariable because of the integration used in the evaluation of the model. These parameters are (1) the difference in the specific growth rates between plasmid-carrying cells and plasmid-free cells (deltamu), and (2) the probability of plasmid loss by plasmid-containing cells (rho(r) mu(+)). The derivative technique used here is completed by mathematical treatments involving data filtering and smoothing. The values of the two parameters are in agreement with those already publised. The current technique does not impose preconditions and permit us to further study related phenomena.     

Effect of growth conditions on the rate of loss of the plasmid pAT153 from continuous cultures of Escherichia coli HB101

Abstract The plasmid pAT153 was lost less rapidly from carbon, nitrogen, phosphorous or sulphur-limited continuous cultures of Escherichia coli HB101 as the dilution rate increased. At a fixed dilution rate of 0.3 h⁻¹, the plasmid was maintained longer as the growth-limiting nutrient was changed from glucose to casamino acids (nitrogen-limited), phosphate or sulphate. These differences in the stability of maintenance were not due to parallel changes in the plasmid copy number. We propose that the rate of loss of pAT153 from E. coli HB101 is determined primarily by the ratio of growth rates of plasmid-containing bacteria and plasmid-free bacteria. This ratio increases with increasing growth rate and depends markedly on the growth-limiting nutrient, sulphate-limited growth being particularly suitable for the maintenance of this host-plasmid combination.     

Isolation of a conjugative plasmid in Escherichia coli determining formaldehyde resistance

A clinical isolate of Escherichia coli which was resistant to the disinfectant formaldehyde was investigated. The strain harboured a plasmid of 62 MDa size. It was shown by conjugation, transformation and plasmid-curing experiments that the formaldehyde resistance is plasmid-mediated and transferable to other strains.     

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence

The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.     

Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains

The occurrence activity and localization of calmodulin in three heterocystous cyanobacteria of the genus Anabaena were studied. Boiled crude extracts caused a Ca2+-dependent stimulation of NAD kinase. Such a stimulation was blocked by EGTA and chlorpromazine, SDS-PAGE and Western blot analysis using antiserum against eukaryotic spinach calmodulin, revealed a polypeptide of about 17 kDa. Immunogold localization of calmodulin gave a dense gold label in both vegetative cells and heterocysts. The label was mainly confined to the centroplasm in vegetative cells, while it was evenly distributed in the cytoplasm of mature heterocysts.     

Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins in Escherichia coli

A pH-inducible promoter system was characterized and its potential applicability in recombinant protein production was evaluated using a plasmid construct, pSM552-545C(-), in which the promoter and activator coding sequences of the cad operon were inserted into the upstream region of a lacZ' reporter gene. Graded gene expression levels with respect to culture pH between 8.0 and 5.5 were observed and the induction range can be as high as 200-fold. The effects of several cultivation parameters, including pH, temperature, induction cell density, and inoculum size, were systematically examined. The practical application of this expression system to high level production of recombinant proteins was successfully demonstrated using a rich medium, superbroth. An extremely high recombinant protein productivity at a value of approximately 1.4 g/L with a specific expression level as high as 35% of total cellular protein can be obtained in a simple batch cultivation. The behavior of this expression system was further investigated using chemostat cultures. An uncommon relationship between the volumetric or specific recombinant protein activity and the dilution rate, with a maximal activity at a dilution rate of approximately 0.4 h(-1)was observed. (c) 1995 John Wiley & Sons, Inc.     

Kinetic modeling of free fatty acid production in Escherichia coli based on continuous cultivation of a plasmid free strain

The microbial production of free fatty acids (FFAs) and reduced derivatives is an attractive process for the renewable production of diesel fuels. Toward this goal, a plasmid-free strain of Escherichia coli was engineered to produce FFAs by integrating three copies of a thioesterase gene from Umbellularia californica (BTE) under the control of an inducible promoter onto the chromosome. In batch culture, the resulting strain produced identical titers to a previously reported strain that expressed the thioesterase from a plasmid. The growth rate, glucose consumption rate, and FFA production rate of this strain were studied in continuous cultivation under carbon limitation. The highest yield of FFA on glucose was observed at a dilution rate of 0.05 h(-1) with the highest specific productivity observed at a dilution rate of 0.2 h(-1). The observed yields under the lowest dilution rate were 15% higher than that observed in batch cultures. An increase in both productivity and yield (≈ 40%) was observed when the composition of the nutrients was altered to shift the culture toward non-carbon limitation. A deterministic model of the production strain has been proposed and indicates that maintenance requirements for this strain are significantly higher than wild-type E. coli.     

High-throughput analysis of the plasmid bioproduction process in Escherichia coli by FTIR spectroscopy

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.     

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

Abstract In an Escherichia coli strain, the levels of penicillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth ratw in chemostat cultures under different nutrient limitation conditions. A considerable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.     

Transformation and expression of a staphylococcal plasmid in Escherichia coli

Abstract A multiple antibiotic-resistant Staphylococcus aureus , was found to possess three plasmid bands in agarose gel electrophoresis. A plasmid of approximately 4.3 kb (pMC790/2) was found to code for ampicillin and tetracycline resistance and to have one Eco RI site when transformed into S. aureus RN 4220. pMC790/2 in unmodified form was transformed into a recA⁻ E. coli at a frequency of 1.2×10⁴ transformants/μg of plasmid DNA. Plasmid (pMC790/2) replicated, maintained itself stably and expressed far better in the E. coli host than in S. aureus .     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: II. Two-stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. (c) 1993 John Wiley & Sons, Inc.     

重组大肠杆菌右旋糖酐蔗糖酶的纯化及其性质

[目的]研究工程菌E.coli BL21(DE3)/pET28-dexYG产右旋糖酐蔗糖酶的纯化和酶学性质.[方法]工程菌经过IPTG诱导后生产含His-tag融合蛋白的右旋糖酐蔗糖酶,通过硫酸铵沉淀、Ni-NTA亲和层析纯化,得到纯度较高的酶蛋白,并对纯酶进行了酶学性质及动力学研究.[结果]经过SDS-PAGE测得该酶的分子量约为170 kDa,与理论推测值基本相同.以蔗糖为底物,酶促反应的最适温度为25～30℃,最适pH值为5.4,动力学常数Km值为10.43 mmol/L;酶活在pH 5.0～8.0较为稳定,在室温(25 ℃)保藏4天仍有59%的酶活力,4℃保存7周酶活力仅下降一半,但在35℃以上失活很快;Ca2 对催化作用有较大的促进,Mg2 有微弱的促进作用,K 对催化反应无影响,Cu2 的抑制作用最强.其他试剂对重组酶的活性有不同程度的影响,其中SDS抑制作用很强.[结论]研究为重组右旋糖酐蔗糖酶纯酶的获取、得到稳定性好、活性高的酶反应体系及利用该酶进行催化反应和工业化应用提供了重要参数.     

Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.     

Factors and markers of virulence in Escherichia coli from human septicemia

A lethal and necrotic factor which causes cell multinucleation in HeLa cell cultures has previously been shown to be coded by the Vir plasmid of Escherichia coli. Using an absorbed rabbit antiserum which neutralized the Vir toxic properties, we have compared the SDS-PAGE immunoblots from laboratory and field strains which either produce or do not produce Vir toxicity. A single band of 110 kDa was found to be specifically associated with vir toxicity in E. coli strains. This antiserum also recognized the 115 kDa protein band which was previously identified as the cytotoxic necretozing factor (CNF) of certain E. coli strains. These results suggest that the toxin coded by the Vir plasmid is a protein of 110 kDa distinct from, but immunologically related to CNF.     

头孢菌素酰化酶基因在大肠杆菌中的表达规律

假单孢菌sp .130头孢菌素酰化酶催化戊二酰 7 氨基头孢烷酸的水解反应 ,生成 7 氨基头孢烷酸。 7 氨基头孢烷酸是医药工业合成大多数头孢菌素衍生物的起始原料。在 6种大肠杆菌表达质粒上构建了表达该酶的不同载体 ,得到了不同表达结果的大肠杆菌转化子。这些质粒有各自的特点 ,适用于不同的场合。     

Adenosine monophosphate-induced amplification of ColE1 plasmid DNA in Escherichia coli

ColE1 plasmid copy number was analyzed in relaxed (relA) and stringent (relA(+)) Escherichia coli cells after supplementation of culture media with adenosine monophosphate (AMP). When a relaxed E. coli strain bearing ColE1 plasmid was cultured in LB medium for 18 h and induced with AMP for 4h, the plasmid DNA yield was significantly increased, from 2.6 to 16.4 mgl(-1). However no AMP-induced amplification of ColE1 plasmid DNA was observed in the stringent host. Some plasmid amplification was observed in relA mutant cultures in the presence of adenosine, while adenine, ADP, ATP, ribose, potassium pyrophosphate and sodium phosphate caused a minor, if any, increase in ColE1 copy number. A mechanism for amplification of ColE1 plasmid DNA with AMP in relA mutant bacteria is suggested, in which AMP interferes with the aminoacylation of tRNAs, increases the abundance of uncharged tRNAs, and uncharged tRNAs promote plasmid DNA replication. According to this proposal, in relA(+) cells, the AMP induction could not increase ColE1 plasmid copy number because of lower abundance of uncharged tRNAs. Our results suggest that the induction with AMP can be used as an effective method of amplification of ColE1 plasmid DNA in relaxed strains of E. coli.     

Temperature shift-up leads to simultaneous and continuous plasmid DNA relaxation and induction of DnaK and GroEL proteins in anaerobically growing Escherichia coli cells

Abstract We previously reported that plasmid DNA in Escherichia coli cells growing under aerobic conditions relaxed immediately and transiently after heat shock (Mizushima, T., Natori, S. and Sekimizu, K., Mol. Gen. Genet. (1993) 238, 1–5). We next examined DNA relaxation and induction of heat shock proteins after heat shock in cells growing under anaerobic conditions. DNA in these cells relaxed rapidly (2 min) after heat shock (42°C), as was the case with aerobically growing cells, but full superhelicity was not recovered. The relaxed state of DNA topology was maintained for 60 min after heat shock. Induction of DnaK and GroEL proteins, which was transient in aerobically growing cells, was continuous in anaerobically growing cells. Therefore, induction of heat shock proteins correlated with DNA relaxation in both aerobic and anaerobic conditions.     

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.