Testing plasmid stability of Escherichia coli using the Continuously Operated Shaken BIOreactor System

Abstract

Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid-bearing and plasmid-free cells. The undesired plasmid-free cells grew 30% faster than the desired plasmid-bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid-bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418-1425, 2016.

Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

“…For the determination of CDWs in batch cultivations, an OD-BTM correlation with a factor of 0.33 was used. The growth rates were calculated according to the method provided in the supplementary material of Sieben et al [ 56 ].…”

The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing

“…From an engineering point of view, higher growth rates seem to be beneficial for RPP as shown in chemostat- and fed-batch experiments ( Vaiphei et al, 2009 ; Slouka et al, 2019 ). Mutations and plasmid loss are expected during long time cultivation of E. coli ( Weikert et al, 1997 ; Sieben et al, 2016 ; Peebo and Neubauer, 2018 ). However, constant supply of antibiotics is believed to prevent plasmid loss in continuous cultures ( Sieben et al, 2016 ).…”

Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli

“…No decrease in viability was observed and kanamycin was continuously fed to the system to avoid plasmid loss. Still, reduced plasmid copy numbers might occur and thus could be an explanation for the decreasing productivity (Sieben et al, 2016). Recent results, however, show that the productivity can fluctuate in lactose induced chemostat with BL21(DE3) as a result of genotypic or phenotypic TABLE 2 | Comparing specific productivity and the product titer at harvest for the production of a cytoplasmic protein; each cycle is investigated separately for fed-batch and repeated fed-batch cultivation; values for chemostat and theoretical "ideal" chemostat cultivations are given every 12 h (i.e., one cycle); calculation for q ; as extracellular protein was found for cultivations of the periplasmic proteins, the secreted protein is shown in dotted lines for the harvested titer; VCC was evaluated by flow cytometry analysis with an average standard deviation of 2%.…”

“…No decrease in viability was observed and kanamycin was continuously fed to the system to avoid plasmid loss. Still, reduced plasmid copy numbers might occur and thus could be an explanation for the decreasing productivity ( Sieben et al, 2016 ). Recent results, however, show that the productivity can fluctuate in lactose induced chemostat with BL21(DE3) as a result of genotypic or phenotypic diversification ( Kittler et al, 2020 ).…”