Working with Plasmids

What are endotoxins

Endotoxins, also called lipopolysaccharides (LPS), constitute the cell membrane elements of gram-negative bacteria, such as E. coli. The outer layer's lipid segment of the bacterial outer membrane is entirely made up of endotoxin molecules (see figure Schematic diagram of the E. coli envelope).

Endotoxins or lipopolysaccharides in bacterial cell wall

A single E. coli cell contains about 2 million LPS molecules, each consisting of a hydrophobic lipid A moiety, a complex array of sugar residues and negatively charged phosphate groups (see figure Schematic diagram of the endotoxin molecule). Therefore, each endotoxin molecule possesses hydrophobic, hydrophilic and charged regions giving it unique features with respect to possible interactions with other molecules. Bacteria shed small amounts of endotoxins into their surroundings while actively growing large amounts when they die. During lysis of bacterial cells for plasmid preparations, endotoxin molecules are released from the outer membrane into the lysate.

Schematic diagram of the endotoxin molecule

Endotoxins significantly reduce transfection efficiencies in endotoxin-sensitive cell lines. Furthermore, endotoxins can influence the uptake of plasmid DNA in transfection experiments by competing with DNA for “free” transfection reagent. Overall, endotoxins represent a non-controllable variable in transfection experiment setup. They are invisible on agarose gels and impossible to detect by optical density and influence the outcome and reproducibility of results and making them difficult to compare and interpret.

Measuring endotoxins

Fun fact:

Historically, endotoxins were measured in a clotting reaction between the endotoxin and a clottable protein in the amoebocytes of Limulus polyphemus, the horseshoe crab.

Today, much more sensitive photometric tests (e.g., Kinetic-QCL Test from BioWhittaker, Inc.) are used, based on a Limulus amoebocyte lysate (LAL) and a synthetic color-producing substrate. LPS contamination is usually expressed in endotoxin units (EU). Typically, 1 ng LPS corresponds to 1-10 EU.

Endotoxin contamination of different plasmid preparation methods

The chemical structure and properties of endotoxin molecules and their tendency to form micellar structures can lead to copurification of endotoxins with plasmid DNA.

On size-exclusion resins, the large size of the micellar form of the endotoxin causes the molecule to behave like a large DNA molecule; and in anion-exchange chromatography, the negative charges present on the endotoxin molecule can interact with anion exchange resins, thus leading to copurification of endotoxins with plasmid DNA.

The level of endotoxin contamination found in plasmid DNA depends on your purification method. QIAGEN Plasmid Kits and Plasmid Plus Kits both yield very pure DNA with relatively low levels of endotoxin. Silica slurry-purified DNA contains significantly higher endotoxin contamination. DNA purified with EndoFree Plasmid Kits contains only negligible amounts of endotoxin (<0.1 EU/µg plasmid DNA) (see table Endotoxin contamination and transfection efficiency using various plasmid preparation methods).

Endotoxin contamination and transfection efficiency using various plasmid preparation methods

Plasmid preparation method	Endotoxin (EU^†/µg DNA)	Transfection efficiency
EndoFree Plasmid Kits	<0.1	154%
QIAGEN Plasmid Plus Kits	<1.0	100%
QIAGEN Plasmid Kits	9.3	100%
Silica slurry	1230	24%

* Host strain: DH5α; plasmid: pRSVcat.

† 1 ng LPS = 1.8 EU.

‡ The transfection efficiency obtained using plasmid prepared with QIAGEN Plasmid Kits was set to 100%. The transfection efficiencies for all other preparation methods were calculated relative to the QIAGEN Plasmid Kit.

Influence of endotoxins on biological applications

How to measure bacterial endotoxins?

In the past, the detection of endotoxins relied on a clotting reaction that occurred when the endotoxin interacted with a clot-forming protein found in the amoebocytes of the horseshoe crab, Limulus polyphemus. Nowadays, highly sensitive photometric assays are used for this purpose. Learn more about endotoxin contamination and removal here.

Removal of endotoxins

Our patented EndoFree plasmid procedure conveniently integrates endotoxin removal into the standard QIAGEN plasmid purification procedure. The neutralized bacterial lysate is filtered through a QIAfilter Cartridge and incubated with a specific endotoxin removal buffer (Buffer ER). The endotoxin removal buffer prevents LPS molecules from binding to the resin in the QIAGEN-tips, allowing purification of DNA containing less than 0.1 endotoxin units per µg plasmid DNA.

Endotoxin-free plasticware and glassware

To avoid recontaminating the plasmid DNA after initial endotoxin removal, we recommend using only new plasticware certified as pyrogen- or endotoxin-free. Pyrogen-free or endotoxin-free plasticware can be obtained from many different suppliers. Endotoxins adhere strongly to glassware and are difficult to remove completely during washing. Standard laboratory autoclaving procedures have little or no effect on endotoxin levels. Moreover, if the autoclave has previously been used for bacteria, the glassware will become extensively contaminated with endotoxin molecules. Heating glassware at 180°C overnight is recommended to destroy any attached endotoxin molecules.

It is also important not to recontaminate the purified endotoxin-free DNA by using reagents that are endotoxin-free. All buffers supplied with EndoFree Plasmid Kits are tested and certified to be endotoxin-free, including the water used to prepare 70% ethanol and the TE buffer for resuspension.