Invitrogen's One Shot Kit
These days, even most of the basic cloning strains are improved with recA and endA mutations to provide increased plasmid yield and quality. The recA gene is responsible for DNA recombination, and strains that contain a mutation of this gene are particularly useful for cloning sequences containing direct repeats. A mutation in the nonspecific nuclease, Endonuclease I (endA), eliminates nonspecific endonuclease activity, resulting in improved plasmid preparations. Stratagene of La Jolla, Calif., now offers many of its most popular strains in a new Gold Series, which incorporates these improvements into the original strains, and also features the Hte phenotype, which contributes to a 100-fold increase in transformation efficiency.
In addition to the plethora of genotypic variations, companies now offer their most popular strains in a choice of either chemically or electro-competent forms and have expanded the choice of packaging formats available. When the diversity of products on the market is not enough, companies such as Invitrogen of Carlsbad, Calif., and Life Technologies, a division of Invitrogen, of Rockville, Md., even offer customized genotype and package format construction according to individual specifications.
|Courtesy of Qbiogene|
A competent Escherichia coli cell
Enhanced Protein Expression
The most popular protein expression system is based on T7 RNA polymerase because of the high level of expression attainable in Escherichia coli. Invitrogen, Stratagene, and Madison, Wis.-based Novagen and Promega, each carry BL21 strains optimized for protein expression. The BL21 strain is naturally deficient in the OmpT and Lon proteases, resulting in a higher yield of intact recombinant proteins. The suffix "DE3" indicates that the host is a lysogen of *DE3, carrying a chromosomal copy of the T7 RNA polymerase gene under the control of the IPTG-inducible lacUV5 promoter. Such strains are used to induce high-level protein expression in T7 promoter-based systems.
Proteins whose overexpression is toxic to E. coli are often difficult to clone, and commonly used high copy number vectors only exacerbate the problem. Since high-level expression of exogenous proteins may be toxic to the cells, some strains also carry a plasmid that encodes for T7 lysozyme (pLysS and pLysE), a natural inhibitor of T7 RNA polymerase. These plasmids prevent leaky expression in uninduced cells by reducing basal expression of the polymerase. Strains carrying pLysS have a lower level of lysozyme expression than do strains carrying pLysE; the availability of both plasmids allows the researcher to fine-tune the level of expression needed.
BL21-SI cells from Life Technologies, a division of Invitrogen, also seek to minimize basal expression via control of T7 polymerase, in this case by driving expression from the salt-inducible proU promoter. The use of this promoter minimizes uninduced T7 polymerase expression, producing improved protein yield and solubility, and enhancing the production of hard-to-express proteins.
Even more refined control of IPTG-induced protein expression is possible using Novagen's Tuner(tm) E. coli strain, which enables true rheostat control of target protein expression levels. The strain is a Lac permease (lacY) deletion mutant that cannot utilize lactose and prevents Lac permease-mediated active transport of IPTG. Since IPTG can also independently enter E. coli, this strain allows for a truly concentration-dependent protein induction simply by adjusting culture concentrations of IPTG.
Stratagene offers ABLE strains as an alternative to cloning a gene into either a low copy number plasmid or a tightly controlled inducible system. The ABLE C and ABLE K strains reduce plasmid copy number four- and 10-fold, respectively, increasing cell viability. The two strains are available for purchase singly or as a kit, allowing the researcher to obtain the highest plasmid copy number still permissive for growth.
Stratagene's CodonPlus series and Novagen's new Rosetta(tm) E. coli cells are designed to enhance the expression of heterologous recombinant genes containing rare codons. Stratagene's CodonPlus competent cells contain extra copies of the genes that encode tRNAs for codons that are rarely used in E. coli and thereby guard against codon bias.2,3 The CodonPlus-RIL cells have extra argU, ileY and leuW tRNA genes for AT-rich genomes, whereas CodonPlus-RP cells have extra argU and proL tRNA genes for GC-rich genomes. In addition, methionine auxotrophs (met) of both series are available for metabolic labeling. Novagen's Rosetta cells are BL21 derivatives that are also designed to enhance expression of eukaryotic proteins containing codons rarely used in E. coli. Therefore, they provide "universal" translation that would be otherwise limited by E. coli codon usage.
Novagen's newest pET system host strains, Origami(tm) E. coli cells, are designed specifically for the expression of active, soluble, properly folded target proteins. These strains carry a double mutation (trxB, gor) in the thioredoxin and glutathione reductases, enzymes in the disulfide bond reductive pathway. These strains are particularly useful for the production of proteins whose solubility is dependent on disulfide bond formation. Novagen also offers the trxB mutation in its AD494 and BL21trxB strains.
Because E. coli cells lack the ability to phosphorylate tyrosine, it was not possible to produce tyrosine- phosphorylated proteins in bacteria before the development of specialized hosts. Stratagene's TKB1 and TKX1 strains carry the elk tyrosine kinase gene controlled by the trp promoter to produce tyrosine-phosphorylated proteins. TKB1 cells are BL21 derivatives equipped for T7 promoter-driven protein expression, whereas TKX1's are XL1-Blue-derived for use with other expression systems.
The BL21Star strain from Invitrogen applies a different approach to improve protein yield. BL21Star strains enhance protein expression and yield by stabilizing mRNA; the cells carry a mutation in the gene encoding RNaseE (rne131), thereby eliminating a major source of mRNA degradation. As a result, more mRNA is available for protein translation.
Life Technologies' Gibco/BRL ELECTROMAX DH10B T1-phage resistant competent cell
Overcoming Difficult Cloning Issues
Few routine laboratory tasks are as tedious and frustrating as screening clones for the presence of an inserted DNA fragment. Researchers may proudly boast of having performed several dozen minipreps to find one precious positive, but doubtlessly, they would have preferred not to have screened so many colonies in the first place. Such situations can be minimized or avoided through the use of *-complementation. Strains containing the lacZM15 marker harbor a mutation of the ß-galactosidase (IacZ) gene in which amino acids 11-41 are deleted. Transformation of these cells with plasmids encoding the missing alpha region of lacZ will restore the gene and enable these cells to utilize lactose, causing them to turn blue when grown on agar plates treated with IPTG and X-gal. If the lacZ alpha region is disrupted by the presence of an inserted DNA fragment, then complementation will not occur, and these colonies will appear white. The popular "Bluescript" vectors from Stratagene are one series of plasmids that can be used in this manner.
Another procedure that is often time-consuming and inefficient is the generation of random gene mutations through PCR or chemical treatment. Mutagenesis procedures are now easier thanks to the development of mismatch repair-deficient mutS strains offered by CLONTECH, of Palo Alto, Calif., Stratagene, and Promega. These strains are unable to carry out mismatch repair, and therefore, do not repair the wild-type/mutant heteroduplexes that are often the result of in vitro site-directed mutagenesis reactions, leading to higher mutation efficiencies. Among the different companies offering mutS strains, the researcher can choose between recA+ or recA- clones, as well as those carrying either kanamycin resistance or sensitivity markers to accommodate different vectors. Stratagene's XL1-Red mutator strain is deficient in all three of the major E. coli DNA repair pathways (mutS, mutD, mutT) and boasts a mutation rate approximately 5,000-fold higher than its wild-type parent.
Most E. coli hosts encode proteins that methylate certain sequences on either adenine or cytosine residues. The Dam methylase modifies the adenine residue in the sequence GATC, whereas the Dcm methylase modifies the second cytosine in the sequence CC(A/T)GG. As a result, DNA obtained from these cells is resistant to digestion with methylation-sensitive restriction enzymes (such as XbaI, which will not digest DNA containing a methylated adenine in the sequence, TCTAGAm6TC). The generation of unmethylated DNA in E. coli is now possible using the dam and dcm strains available from Invitrogen, Stratagene, and Carlsbad, Calif.-based Qbiogene.
Likewise, the cloning of methylated DNA has never been easier. E. coli strains methylate their DNA as a way of identifying foreign nucleic acids (strain K methylates adenosine residues in the sequence AA(N)6GTGC, whereas strain B methylates adenosines in the sequence TGA(N)8TGCT). Since eukaryotic genomic DNA can be highly methylated in a pattern dissimilar to that of E. coli, it is often cleaved by host restriction systems. Such cleavage prior to host replication creates libraries lacking complete representation. Strains carrying E. coli K12 restriction system mutations such as mcrA, mcrCB, mrr and hsd, which prevent the cleavage of cloned DNA from non-E. coli sources, reduce the degradation of transformed DNA in vivo. The genotypes of strains containing these mutations will usually indicate whether both the restriction and methylation (r-,m-) activities have been inactivated, or only the restriction (r-,m+) activity.
Another useful genotype is the deoR- mutation. Strains with this genotype efficiently take up large DNA molecules, assisting in library construction. In addition to the better-known high-efficiency strains, Invitrogen has introduced GeneHogs, a DH10B-derived E. coli strain specially prepared to take up large DNA such as bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs).
Some eukaryotic genes contain unstable inverted repeats or secondary structures, such as Z-DNA, that can be destroyed by host repair systems. The deletion of E. coli genes involved in DNA rearrangement and deletion has helped in the cloning of such unstable DNA. Stratagene's SURE competent cells have deletions in both the UV repair system (uvrC) and the SOS repair pathway (umuC) resulting in a 10- to 20-fold higher stability of DNAs containing long inverted repeats. Removal of genes encoding the SbcC, RecJ, and RecB proteins involved in recombination increases Z-DNA stability and reduces homologous recombination.
As reported here recently,4 Life Technologies, a Division of Invitrogen, offers MAX Efficiency DH5* T1 Phage-Resistant Competent Cells. This T1 phage-resistant strain of E. coli is particularly useful for researchers and organizations worried about the safety and security of precious bacterial stocks and libraries.
Suppliers of Competent Cells
Life Technologies,a Division of Invitrogen Corp.
Easier than Ever
Large, bulk aliquots are no longer the only option for purchasers of competent cells; a number of companies now offer a variety of purchasing formats. For example, Invitrogen's One Shot Kits and Novagen's Singles offer the ultimate in transformation convenience and efficiency. The single-use design of these products provides competent cells in reaction-ready 50-µl pre-aliquoted tubes. Cells are thawed just once so they are not subjected to the repeated freeze-thaw cycles that compromise efficiency, and the transformation reaction can be carried out directly in the tube in which the cells arrive. These kits can be ordered in different sizes depending on the number of aliquots needed.
On the other end of the spectrum are high-throughput needs, for which neither bulk aliquots nor single-tube formats are adequate. As the human genome sequencing project and pharmaceutical industry identify thousands of sequences as potential therapeutic targets, several companies are taking advantage of the need for a more broad-scale approach for scientists studying protein function. Novagen's HT96(tm) competent cells are designed for high-throughput cloning and protein expression. The cells arrive predispensed in 20-µl aliquots in a 96-well polypropylene plate compatible with a variety of thermal cyclers and water baths for performing the transformation reaction. This format can be scaled down as needed, as individual tubes or groups of wells can be cut from the whole plate for processing a smaller number of samples. Likewise, Invitrogen has an optimized 96-well format for high-throughput applications in its MultiShot design. Currently, the company's TOP10 cells are available in the 15-µl-aliquot/well MultiShot format or in a newer MultiShot StripWell design. Instead of a 96-well microtiter plate, the MultiShot StripWell contains 96 tubes with 8 tubes per strip; the tubes within the strips can be snapped off easily for convenience. Stratagene offers its high-efficiency XL10-Gold strain in a comparable 96-well format optimized for convenience and high-throughput needs.
The vendors listed in the accompanying table supply a wide range of competent cell strains, with varying genotypes, package sizes, and transformation efficiencies. When examining the genotypes of these strains, note that it is customary to list only those genes that are mutant, not wild-type. Thus a strain that is, for example, dam dcm, can produce neither the Dam nor Dcm methylase. However, genes listed as being present on the F' episome or another plasmid, are wild-type unless otherwise indicated. S
Barbara A. Cunningham (Barbara.A.Cunningham@Hitchcock.org) is a freelance writer in Lyme, N.H.
2. C.-P. Carstens, A. Waesche, "Codon bias-adjusted BL21 derivatives for protein expression," Strategies, 12:49-51.
3. D. Swanson, "Unbiased," The Scientist, 14:22, May 29, 2000.
4. A.L. Francis, "Combating phage rage," The Scientist, 14:23, Oct. 2, 2000.