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Introduction to ClearColi® technology:

Is there a better way to eliminate endotoxin contamination?
Now there is. 

Instead of removing lipopolysaccharide (LPS) contamination from your protein or plasmid DNA preparations, eliminate the LPS at the source. Genetically modified LPS from a novel E. coli strain produces functionally clean recombinant proteins and plasmids.  ClearColi® cells are the first commercially available competent cells with a modified LPS (Lipid IVA - see Fig. 1) that does not trigger the endotoxic response in mammalian cells. ClearColi cells lack outer membrane agonists for hTLR4/MD-2 activation; therefore, activation of hTLR4/MD-2 signalling by ClearColi is several orders of magnitude lower compared with E. coli wild-type cells, and plasmid DNA prepared from ClearColi is virtually free of endotoxic activity. After minimal purification from ClearColi cells, proteins or plasmids (which may still contain  Lipid IVA ) can still be used in most applications without eliciting an endotoxic response (see Endotoxicity LAL Levels for details).

Figure 1. The LPS of a normal E. coli cell compared to the genetically modified Lipid IVA from ClearColi cells.  In ClearColi, the oligosaccharide chain has been deleted, and two of the six acyl chains have been removed to disable the endotoxin signal.

Modifications to the genotype of the ClearColi cells consist of seven separate gene deletions, thereby ensuring that there is no chance of genetic reversion back to wild type and production of normal LPS.  These mutations result in the deletion of the oligosaccharide chain from the LPS, making it easier to remove the resulting lipid IVA from the downstream product.  More importantly, two of the six acyl chains are deleted.  The six acyl chains of the LPS are the trigger that is recognized by the Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causing activation of NF-?B and production of proinflammatory cytokines.  Lipid IVA, which contains only four acyl chains, is not recognized by TLR4 and thus does not trigger the endotoxic response (see Fig. 2).

Fig. 2. Comparison of relative NF-κB induction in HEK-Blue Cells using purified LPS from a K-12 E. coli strain or from pure, synthetically manufactured Lipid IVA.

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Genotype Information:

ClearColi K-12 competent cells have the following genotype:

F-, λ- ΔendA ΔrecA msbA52 frr181 ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA

Seven specific deletion mutations (ΔgutQ ΔkdsD ΔlpxL ΔlpxMΔpagPΔlpxPΔeptA) encode the modification of LPS to Lipid IVA, while one additional compensating mutation (msbA52) enables the cells to maintain viability in the presence of the LPS precursor lipid IVA.

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Why Endo-free Plasmid Prep is not the Best Method

To prevent toxicity in cells to be transfected, plasmids produced in E. coli must be essentially free of endotoxin. However, efficient elimination of endotoxin is a challenging task, and endo-free plasmid prep methods are expensive and time consuming.  ClearColi K-12 cells produce plasmid DNA with endotoxin levels less than or equal to plasmids prepped from standard E. coli cloning lines and Qiagen's Endofree Maxi Prep kits. 

ClearColi K-12 cells allow use of standard plasmid prep instead of endo-free methods:

• Saves up to 90% in plasmid prep costs
• Saves 1 hour or more in prep time
• High transfection and protein expression levels without concern for endotoxin contamination

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Plasmid Production with ClearColi K-12 Cells

ClearColi K-12 cells are endA- and recA- for the highest quality plasmid production.  Plasmid yields from ClearColi K-12 cells are equal or greater than those obtained from normal DH10B competent cells.  Table 1 compares yields from 1 mL minipreps for both ClearColi K-12 and E. Cloni 10G (equivalent OD's were used for both preps). 

	Plasmid DNA Yield  (standard miniprep)
ClearColi K-12	4.83 µg/mL
E. Cloni 10G	3.75 µg/mL

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Endotoxicity/LAL Levels

Limulus amebocyte assay testing is an FDA-approved method for detection of endotoxins and the most common assay used.  As shown in figure 3, a standard plasmid purification step for DNA produced from ClearColi cells results in LAL response levels less than 1% of that produced by plasmids derived from standard DH10B cells and standard prep methods.  The EU levels detected from ClearColi K-12 derived plasmids are also equivalent or lower than to those obtained from DH10B derived plasmids prepared with Qiagen's Endofree Maxi prep kits (data not shown).

Fig. 3 Comparison of post-plasmid purification endotoxin units detected from ClearColi K-12 (red bars) and DH10B (E. cloni 10G, grey bars) competent cells. Plasmid DNA from ClearColi demonstrates significant reduction in EU/mg without the need for endotoxin free plasmid prep kits.

It should be noted that the residual EU measurements are likely due to the non-specific nature of the LAL assay unless extraneous LPS contamination from other sources is present. The LAL assay is activated solely by the4´-monophosphoryldiglucosamine backbone of LPS.  LAL activity is minimally influenced by acylation pattern of LPS, the key determinant of endotoxicity in eukaryotic cells.  The LAL assay also recognizes a wider spectrum of LPS/lipid A variants than the central cellular endotoxin sensor system of the human immune cell system.  As such, false positive results can and will result due to the lack of specificity of the assay.

Alternative toxicity assays, such as those using HEK-Blue cells (see ClearColi® BL21(DE3) cells for more information) suggest that even in the presence of EU levels above threshholds normally targeted by researchers, the actual immunogenic effects from ClearColi-derived products are non-existent.  Due to the non-specificity of the LAL assay when combined with lipid IVA from ClearColi, it is suggested that researchers consider alternative methods of endotoxin measurement.

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Transfection and protein expression from ClearColi Plasmids

With the original source of endotoxin eliminated, it is now possible to transfect plasmid DNA prepped with standard methods directly into human or other mammalian cell lines without concern for cell viability, altered cellular responses or poor protein expression.  To prove this, a plasmid (pME-HA) containing a gene encoding a fluorescent protein was cloned into both ClearColi K-12 and DH10B E. coli.  The plasmid from ClearColi was then isolated via standard Qiagen Maxi prep kit method, while the plasmid from DH10B was isolated using Qiagen's Endofree Maxi Kit.  The resulting plasmids were transfected into HEK293T cells for protein expression (Figure 4).   No differences in cell viability or protein expression levels have been observed when using a non-endofree plasmid prep method in combination with ClearColi-derived plasmids. 

Figure 4. Comparison of expression of a green fluorescent protein in HEK293T cells from ClearColi-derived plasmids and standard maxi prep (left) vs. DH10B-derived plasmids and endofree maxi prep (right). The upper panels show fluorescence; the lower panels show a combined fluorescence and bright field image.

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Growth Rates for ClearColi K-12 Cells

ClearColi K-12 cells grow at approximately 50% of the rate of normal DH10B cells (see Fig. 5).  Users should expect to see very small colonies for the first 24 hours after plating transformants.  Lucigen recommends incubating plates for 32-40 hours before picking colonies for future experiments.  When grown to sufficient densities, ClearColi K-12 cells produce similar plasmid yields as normal DH10B cells.

Figure 5. Comparison of growth rates for ClearColi K12 Electrocompetent Cells vs. E. cloni 10G ELITE Electrocompetent cells. Cells were transformed with pME-HA-Comet and inoculated to an initial OD600 of ~0.01 in 200 mLof LB Miller medium and grown at 37° C with shaking at 210 rpm. The OD600 of the cultures was recorded every half hour.

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Relevant Reference Articles:

• Teghanemt, et al, Molecular Basis of Reduced Potency of Underacylated Endotoxins, J Immunol, 2005;175:4669-4676
• Mamat, et al, Single amino acid substitutions in either YhjD or MsbA confer viability to 3-deoxy-D-manno-oct-2-ulosonic acid-depleted Escherichia coli, Molecular Microbiology, 2008, 67(3), 633–648
• Meredith, et al, Redefining the Requisite Lipopolysaccharide Structure in Escherichia coli, ACS Chemical Biology, 2006, 1(1), 33-42
• Brandenburg, et al, The Expression of Endotoxic Activity in the Limulus Test as Compared to Cytokine Production in Immune Cells, Current Medicinal Chemistry, 2009, 16, 2653-2660
• Gutsmann, et al. Structural prerequisites for endotoxic activity in the Limulus test as compared to cytokine production in mononuclear cells, Innate Immunity, 2010, 16(1), 39-47
• Beom Seok Park1 et al., The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex,Nature 458, 1191-1195 (30 April 2009)

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ClearColi Licensing Information:

ClearColi Competent cells are subject to US Patent 8,303,964 and other US and foreign pending patents.

Lucigen Corporation ("Lucigen") has a license from Research Corporation Technologies to sell ClearColi competent cells to third-parties for non-commercial research purposes only. A separate license is required for any commercial use. For more information about the use of this product by commercial entities, please review our full licensing page.

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