Abstract Experimental elucidation of the metabolic load placed on bacteria by expression foreign protein is presented. The host/vector system Escherichia coli RR1/pBR329 (amp r , cam and let ). Plasmid content results, which indicate that plasmid copy number monotonically increases with decreasing growth rate, are consistent literature ColE1‐like plasmids. More significantly, we have experimentally quantified reduction in rate brought about chloramphenicol‐acetyl‐transferase (CAT)...

Cybernetic models, developed earlier by the authors, have been evaluated experimentally for growth of Klebsiella oxytoca in batch cultures using mixed substrates from glucose, xylose, arabinose, lactose, and fructose. Based entirely on information procured single substrates, models accurately predict without further parameter fitting, diauxic automatically predicting order which are consumed. Even triauxic a mixture lactose is predicted model based substrate data. Growth glucose-fructose...

Abstract The internal regulatory processes, which underlie a variety of behavior in microbial growth on multiple substrates, are viewed as manifestation an invariant strategy to optimize some goal the cells. A goal‐seeking or cybernetic model is proposed here, with optimization obased short‐term perspective response environment. parameters determined from data single substrates. predicts entire range substrates simultaneous utilization all sugars sequential pronounced diauxic lags. It shown...

A structured kinetic model is developed to describe the dynamics of hybridoma growth and production monoclonal antibodies metabolic waste products in suspension culture. The crucial details known processes cells are incorporated by dividing cell mass into four intracellular pools. framework structure allow dynamic calculation instantaneous specific rate a steady state simulations equations exhibit excellent agreement with experimentally observed trends substrate utilization product...

Abstract The continuous separation of nonviable hybridoma cells from viable by using a narrow rectangular channel that is inclined the vertical has been investigated experimentally. effectiveness settler in selectively retaining hybridomas bioreactor while permitting removal shown to depend on flow rate through settler. Intermediate rates have found provide highest relative retention. At high dilution chemostat, over 95% could be partitioned bottom 50% are removed top This successful due...

Abstract Several published experiments in the literature have provided main guidelines for a quantitative modeling approach called “cybernetic modeling.” Cybernetic models correlated bacterial growth with mixed substrate systems remarkable accuracy.

The instantaneous specific growth rate of a recombinant bacterial culture is directly calculated using simple structured kinetic modeling approach. Foreign plasmid replication and foreign protein expression represent metabolic burdens to the host cell. individual effects these plasmid-mediated activities on plasmid-bearing cells are estimated separately. dynamic steady state simulations model equations show remarkable agreement with widely observed experimental trends in copy number content....

Abstract The perfusion culture of suspended mammalian cells requires a cell retention device, the best which will retain all viable and reject nonviable debris. inclined settler is passive, simple, inexpensive, easy‐to‐maintain device that has been shown in past to selectively remove single hybridoma cultures. In this work, we have demonstrated preferential return recombinant Chinese hamster ovary (CHO) through use three‐port maintained at lower temperatures vibrated reduce attachment...

The dynamics of chemically induced chloramphenicolaceyl-transferase (CAT) expression are determined in batch cultures Escherichia coli DH5alphaF'IQ [pKK262-1]. This article is directed towards understanding the coupling cloned-protein synthesis and reduced cell growth which balanced optimal system. Experimental results indicate that best inducer (IPTG) concentration near 1.0 mM when added during midexponential growth. Lower concentrations cause only weak induction, whereas higher...

Abstract Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting the native glycolytic Entner Doudoroff (ED) has been hypothesized. We have investigated this putative reaction by developing a kinetic model incorporating all PP and ED pathways, including those catalyzed enzymes. Starting experimental literature on in vitro characterization each...

Abstract Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation intracellular foreign protein in single during cycle Chinese hamster ovary (CHO) transfected with an vector containing dihydrofolate reductase ( dhfr ) and lacZ bacterial β‐galactosidase (a nonsecreated protein). The was under control constitutive cytomegalovirus promoter. These normally attachment‐grown...

A novel Eschericha coli expression system directed by bacteriophage T7 RNA Polymerase utilized for overexpression of the cloned gene. The recombinant cell contains plasmid with a promoter, to regulate target This promoter is recognized only polymerase, whose gene has been fused into host chromosome and under control lacUV5 promoter. Therefore, on can be expressed in presence which induced isopropyl-beta-D-thiogalactopyranoside (IPTG). batch cultures were performed investigate effect...

We have investigated the independent effects of selective gene amplification (using dhfr amplifiable selection marker) and culture operating strategy (batch vs repeated fed-batch semicontinuous perfusion) on glycosylation a recombinant reporter protein (secreted alkaline phosphatase, SEAP) produced in transfected Chinese hamster ovary (CHO) cells. HPLC analyses coupled with susceptibility to various exoglycosidases were used determine N-glycosylation profile SEAP samples. The amplified cell...

Large-scale cultivation of murine bone marrow cells was accomplished in an airlift packed bed bioreactor system designed to mimic the vivo environment. The attachment-dependent stromal cell population, which provides necessary microenvironment, including growth factors for subsequent hematopoietic activity, first established within bioreactor. This occurred on fiber-glass matrix annular region Once layer established, fresh were inoculated initiate hematopoiesis. However, traditional culture...

The primary advantage of an inducible promoter expression system is that production the recombinant protein can be biochemically controlled, allowing for separation unique growth and phases culture. During phase, culture rapidly grown to high cell density prior induction without extra metabolic burden exogenous production, thus minimizing nonproductive period Induction at ensures volumetric will maximized. In this work, we have demonstrated feasibility overexpressing a reporter glycoprotein...