Stable Expression System Introduction

stable-mammanlian-cellMammalian expression system into a cell can be transient or stable. stable expression system can refer to a cell line that is simply stable, in that it will remain unchanged over a long time. Not a common usage of the term. A cell line by definition is pretty stable, at least most are. It can refer to a cell line that has been transfected or infected with a foreign piece of DNA that has incorporated itself into the genome of the cell.

From: https://answers.yahoo.com/question/index?qid=20101026025934AAandlo

In stable expression system, the plasmid DNA is integrated into the chromosomes, or as an episome a separate piece of nuclear DNA), and gets passed on to future generations of the cell. This is a much rarer occurrence and complex to perform, as sometimes only a portion of the plasmid gets integrated, which may not contain the gene of interest. All stable expression system transfections start out as a transient transfection, with selectable markers that are able to distinguish any cells that have successfully integrated the gene into their genome. A common method used is to co-transfect the gene of interest with another gene for antibiotic resistance, and treat the transfected cells with the antibiotic. Repeating antibiotic treatment for long-term cell cultures results in the expansion of stably-transfected cells. Linear DNA appears to be better for stable expression system, although its uptake is lower than supercoiled DNA.

From: http://www.cell-transfection.com/transient-versus-stable-transfection/

Overview of Mammalian Expression System

mammalian cell transfectionMammalian expression system are generally capable of producing larger amounts of properly folded secreted proteins with usually correct glycosylation pattern for mammalian or other eukaryotic proteins. In case of cytoplasmic proteins or multi-protein complexes, mammlian expression system show usually lower expression levels than insect cells which are also capable of secreting proteins if a native-like glycosylation pattern is not essential for the functionality of the target protein or the planned down-stream application. Mammalian cells expression system covering transient expression system and stable cell line expression system.
from:https://www.embl.de/pepcore/pepcore_services/protein_expression/

Mammalian expression system

Transient transfection

Transiently transfected cells express the foreign gene but do not integrate it into their genome. Thus the new gene will not be replicated. These mammalian cells express the transiently transfected gene for a finite period of time, usually several days, after which the foreign gene is lost through cell division or other factors.

Stable transfection

Generating stably transfected cells begins with a transient transfection, followed by an infrequent but important and serendipitous process. In a small proportion of transfected cells, the foreign gene is integrated into the cells’ genome. The hallmark of stably transfected cells is that the foreign gene becomes part of the genome and is therefore replicated. Descendants of these transfected cells, therefore, will also express the new gene, resulting in a stably transfected cell line.

As the development of stably-expressing cell lines can be laborious and challenging, the ability to use transient expression when appropriate is beneficial. Along with new transfection techniques will undoubtedly come the further refinement of both transient and stable transfection methods, enabling researchers to accomplish gene expression tasks as efficiently as possible.

Mammalian expression system category from:
http://www.biocompare.com/Editorial-Articles/126324-Transfection/

Bacterial Fermentation Definition

E.coli Protein Expression Service

Bacterial Fermentation Definition

bacterial fermentation definitionMost energy-conserving reactions in living organisms are redox reactions. One substrate is oxidized with the concomitant reduction of another substrate. In chemoorganotrophic aerobes, the substrate reduced is usually oxygen. In respiring anaerobes, the electron acceptor can be either organic or inorganic. Typical examples are the sulfate-reducing or methanogenic organisms (carbon dioxide). In respiring organisms, both aerobic and anaerobic, most of the energy is produced by electron transport phosphorylation. This is in contrast to fermentations, in which most of the adenosine triphosphate (ATP) is synthesized by substrate level phosphorylation.

Fermentation is an anaerobic redox process, in which the oxidation of the substrate is coupled to the reduction of another substrate or an intermediate derived from the oxidation, with the difference in redox potential of the substrate and the end product providing energy for ATP synthesis. In most fermentations, the same substrate is used as both reductant and oxidant, whereas in some amino acid fermenting organisms, one amino acid is oxidized and another is reduced (Stickland reaction). The oxidation reaction is coupled to substrate level phosphorylation whereas the reduction reaction is usually not. The fermentation end products are excreted. The nature of these products is different in various species, and the various fermentation pathways are named after their main products.

High Cell-Density Fermentation of Escherichia coli

This is a 5-day protocol for high cell-density bacterial fermentation and overnight over-expression of (a) target protein(s) using a computer controlled fed-batch procedure.

Protocol in short  
Day 1 Assembly of the fermenter vessel Preparation and autoclavation of the fermenter vessel

  • Preparation for autoclaving
  • Calibration of the pH probe (external)
  • Autoclaving the fermenter vessel

Preparation of the starter culture

Day 2  Preparation of inoculation cultures Preparation of the fermenter vessel for cultivation

  • Polarizing the DO probe (for at least 6 hours)
  • Recalibration of the pH probe (internal)
  • Setting the fermentation parameters
  • Calibration of the DO probe

Inoculation of the fermenter vessel and start of the batch phase

Day 3 Start of the fed-batch phase

  • Start of the feeding protocol

Induction of protein expression

Day 4  Harvesting of the cells

  • Harvesting the cells
  • Cleaning the feed tubing
  • Autoclaving the fermenter vessel
Day 5

Cleaning of the fermenter vessel

More details for experimental steps: High cell-density fermentation of Escherichia coli

The BIC(BiologicsCorp) bioprocess facility provides services in microbial fermentation. It features a 1,000 sq. ft. pilot scale fermentation laboratory capable of handling class II organisms, an adjoining 500 sq. ft. bench scale cell culture, and fermentation laboratory with multiple fermenters and bioreactors for microbial. The facility can manufacture recombinant proteins and metabolites, as well as media optimization and cell culture scalability experiments. The fermentation facility provides clients with seed culture development and culture scale-up from shake flasks to pilot scale fermenters.

Microbial Fermentation Process

fermentation-process

BIC(BiologicsCorp) has a number of 5L, 50L, 100L, and 1000L fermentors available on-site for fermentation runs. BIC’s fermentation services include:

  • 5 L to 1000 L fermentors for process development
  • High cell density fermentations
  • Batch, fed-batch and perfusion capabilities
  • Continuous flow centrifuge & hollow fibers for separation of biomass from broth
  • Fermentation optimization