ClearColi Competent Cells – Frequently Asked Questions:
(from the Lucigen website)
Q: What is the difference between ClearColi competent cells and other commercially available cells?
A: ClearColi competent cells have a genetically modified lipopolysaccharide (LPS) that disables the trigger for endotoxic response in human cells. This has been accomplished by incorporating seven genetic deletions (ΔgutQ ΔkdsD ΔlpxL ΔlpxMΔpagPΔlpxP ΔeptA) that modify LPS to lipid IVA, while one additional compensating mutation (msbA148) enables the cells to maintain viability in the presence of the LPS precursor lipid IVA.
Q: What is the difference between LPS and lipid IVA?
A: In ClearColi cells, the two secondary acyl chains of the normally hexa-acylated LPS have been deleted, which is a key determinant of endotoxicity in eukaryotic cells. The six acyl chains of the LPS are the trigger which is recognized by the Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causing activation of NF-κB and production of proinflammatory cytokines. The deletion of the two secondary acyl chains results in lipid IVA, which does not induce formation of the activated heterotetrameric hTLR4/MD-2 complex and thus does not trigger the endotoxic response. In addition, the oligosaccharide chain is deleted, making it easier to remove the resulting lipid IVA from any downstream product.
Q: I measured endotoxin levels via LAL assay, and I’m still obtaining a positive response. Why?
A: Limulus amebocyte lysate (LAL) assay testing is an FDA-approved method for detection of endotoxins and the most common assay used. However, the LAL assay is an inappropriate method to discriminate between endotoxically active hexa-acylated LPS and endotoxically inactive tetra-acylated lipid IVA. The structural requirements for activation of the LAL cascade by endotoxins differ from those for stimulation of the human immune cell system. While the acylation pattern of LPS/lipid A is a key determinant for stimulation of human immune cells, activation of the LAL cascade only marginally, if at all, depends on the number of acyl chains. Instead, reactivity in the LAL assay requires the 4´-monophosphoryl-diglucosamine backbone structure, which is present in both hexa-acylated LPS and tetra-acylated lipid IVA of E. coli. As such, false positive results are due to the lack of specificity of the assay. The LAL assay recognizes a wider spectrum of LPS/lipid A variants than the central cellular endotoxin sensor system of the human immune cell system.
A simple Ni-column purification step for proteins produced from ClearColi cells significantly reduces LAL response levels. For example, Lucigen demonstrated a 99% reduction in LAL response comparing ApoA1 expressed using ClearColi cells versus E. cloni® EXPRESS BL21(DE3) Electrocompetent Cells.
Cell Line LAL Results (EU/mg) Percent Reduction ClearColi Electrocompetent Cells 450 99.1% E. coloni EXPRESS BL21(DE3) Electrocompetent Cells 53800 99.1%
Residual EU measurements are due to the non-specific nature of the assay unless extraneous LPS contamination from other sources is present. Alternative endotoxicity assays, such as those using HEK-Blue™-4 cells (InvivoGen) suggest that even in the presence of EU levels above normal thresholds targeted by researchers, the actual stimulating effects from ClearColi-derived proteins are non-existent.
Due to the non-specific nature of the LAL assay giving false positive endotoxic results with lipid IVA from ClearColi, it is suggested that researchers consider alternative physiologically relevant methods.
Q: What levels of protein expression can I expect from ClearColi BL21(DE3) cells?
A: When grown to sufficient densities, ClearColi BL21(DE3) cells produce similar protein levels as normal BL21(DE3) cells when comparing equal numbers of cells. Some variation of expression levels may be seen with different proteins.
Q: Are recombinant proteins expressed from ClearColi BL21(DE3) cells soluble and functional?
A: Lucigen has expressed soluble, functional fluorescent proteins from ClearColi BL21(DE3) cells. No significant difference between normal BL21(DE3) and ClearColi BL21(DE3) cells is expected with regards to solubility or functionality.
Q: Are ClearColi cells really endotoxin free in mammalian cells?
A: Lipid A from ClearColi cells is modified such that it is incapable of inducing an endotoxic response in human immune cells. Seven separate deletions ensure that it cannot revert to normal LPS production. With proper controls, plasmids and proteins can be produced from ClearColi cells without need for downstream endotoxin removal steps. However, LPS contamination may be prevalent in your laboratory, and care must be taken to minimize LPS sources other than your cell strain.
While lipid IVA is known as an endotoxin antagonist in human LPS-responsive cells, it has to be taken into account that the tetra-acylated LPS precursor may act as an endotoxic activator in other mammalian hosts such as mouse, Chinese hamster or equine cells, which reflects animal species-specific recognition and stimulatory activity of lipid IVA due to species-specific differences in the structures of TLR4 and MD-2.
Q: How do I avoid contaminating my proteins or plasmids with endogenous LPS?
A: Good laboratory sterile technique can adequately control LPS contamination from extraneous sources. Lucigen recommends the following precautions.
- Use disposable pipette tips and centrifuge tubes certified as sterile and non-pyrogenic
- Depyrogenate any glassware by heat treating at >250° for 1 hour prior to use
- Do not use purification columns or resins that have come in contact with normal E. coli cell strains
- Use reagents certified as low endotoxin or test reagents prior to use
- Use a water source that is regularly tested for endotoxin contamination
- Clean all laboratory surfaces with disinfectants
Q: Do I need to measure endotoxins after purification if I use ClearColi?
A: In applications where minimal endotoxin levels are critical, Lucigen strongly recommends all normal precautions are taken. We cannot guarantee a total absence of LPS due to the possibility of contamination from other sources. Safety and downstream applications are the sole responsibility of the user.
Q: Is endotoxin removal after purification necessary?
A: This will depend on the user’s method of endotoxin measurement and application. As previously discussed, the lipid IVA of ClearColi cells does not cause an endotoxic response in human cells; however, the use of LAL testing may result in a relatively low EU measurement. Normally, a simple plasmid purification or Ni-column protein purification will be sufficient to lower LAL levels below threshold. If lower levels are desired, additional cleanup steps should be taken.
Q: What is the growth rate of ClearColi BL21(DE3) cells?
A: ClearColi BL21(DE3) cells grow at approximately 50% of the rate of normal BL21(DE3) cells. 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. Longer growth times are necessary to reach desired cell densities prior to inducing protein expression.
Q: What are the expected colony characteristics of ClearColi BL21(DE3) cells?
A: Some variation in colony size may be observed when plating ClearColi cells. A small portion (<2%) of colonies may be larger than the general population. In general, these larger colonies have shown protein expression and endotoxin levels similar to the typical size colonies. Lucigen recommends the use of typical size colonies for protein expression. For best results, freshly transformed colonies should be used and multiple passages should be avoided.
Q: It is well known that genetic lesions leading to truncation of the LPS molecule are associated with alterations of bacterial cell envelope properties. How does the expression of lipid IVA affect cell envelope properties, outer membrane integrity and cell viability of ClearColi?
A: The genotype of ClearColi cells not only blocks biosynthesis of hexa-acylated lipid A but also prevents glycosylation of the lipid IVA precursor, resulting in the deepest LPS mutant possible and an outer membrane of correspondingly compromised integrity. The lack of any glycosylation has the consequence that the envelope of ClearColi cells is extremely hydrophobic. As a result, the ClearColi cells tend to aggregate, so rapid sedimentation of the cells without shaking of the suspensions is a usual phenomenon. We therefore recommend shaking ClearColi cells vigorously in flasks or tubes to avoid settling out during growth. Furthermore, always vortex the cells for further use, e.g. preparation of serial dilutions.
ClearColi cells are prototrophs and carry no antibiotic resistance markers. However, the cells show a salt-dependent growth defect in hypotonic media, which suggests that outer membrane instability of ClearColi increases under conditions of low ionic strength. For good growth and to avoid production of the colanic acid exopolysaccharide as a stress response, ClearColi requires 1% NaCl in media. Use LB-Miller but not LB-Lennox medium.
ClearColi can be stored without any problems as glycerol stocks at –80°C in LB-Miller medium containing 20% glycerol. However, we recommend to always stock ClearColi cells immediately after strain construction experiments, e.g. plasmid transformations. We do not recommend storing ClearColi strains on agar plates at 4°C for more than a week. While the cells remain viable, there is an increased lag time before growth.