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Production of Recombinant Small Interfering RNA using E-coli by high cell density Fed-batch fermentation in MINIFOR fermenter
Reference: G. Kaur, H‐C. Cheung, W. Xu, J.V. Wong, F.F. Chan, Y. Li, L. McReynolds, L. Huang “Milligram scale production of potent recombinant small interfering RNAs in Escherichia coli”, Biotechnology and Bioengineering. 2018;1–12.
Small interfering RNAs are used in gene silencing to study gene functions, treatment of cancer and other diseases. To produce a recombinant small interfering RNAs in Escherichia coli, a research was carried out in the City University of Hong Kong. They achieved milligram scale production of pro‐siRNA in MINIFOR bioreactor and a yield improvement by over 200‐fold compared with the traditional shake flask culture method.
Different recombinant siRNA clones of E.coli were cultivated in the MINIFOR Fermenter to study siRNA production efficiency. High cell density fermentation was performed in Batch cultivation followed by Fed-batch fermentation. Results obtained in fed‐batch process showed that optical density (OD) of an average culture at 600 nm were 39.8 and 41.7 g/L biomass (wet cell weight). With a high–cell density fed‐batch fermentation method in the MINIFOR Bioreactor, the optimized clone produced a yield of ~10 mg purified pro‐siRNA/liter of bacterial culture.
Selection of best clones that are producing your product of interest is a time-consuming process, especially if the optimization and selection performed in a fermenter. Cleaning, preparation, setting up a fermenter for Autoclave before a Run is a difficult job with traditional fermenters. But these problems are eliminated in the MINIFOR Fermenter and Bioreactor system by introducing many revolutionary changes.
MINIFOR is advantageous to work with aerobic and micro-aerophilic or anaerobic bacterial (e.g.: E. coli, B. subtilis, B. anthracis, B. amyloliquefaciens, B. pumilus, B. licheniformis, S. aureus, etc.) and yeast (e.g.: S. cerevisiae, P. pastoris, S. pastorianus, S. uvarum, etc.) cultures (co-culture) under batch, fed-batch or continuous (chemostat) mode of operation. Parameter control: temperature, agitation, pO2 (OD), pH, air flow rate by internal thermal MASSFLOW gas flow control (controlled supply of air for aerobic or N2 for anaerobic process)
The high quality LAMBDA peristaltic pumps have been specially developed for long term continuous processes like fermentations and cell cultures.
LAMBDA's unique tubing compressing mechanics reduces pulsation and is very gentle to the tubing. No clamps, fixings or stoppers are required to hold the tubing in place. This leads to a long tubing life and the long term stability of flow rates even with low cost tubing.
Features of LAMBDA MINIFOR bench-top fermenter could be found at www.fermentor.net/features and www.lambda-instruments.com/fermenter-bioreactor/
Keywords: E.coli fermentation, Batch, Fed-batch, High cell density fermentation, Small interfering RNA production, Fermenter.
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