Using synthetic biology methods, the Escherichia coli K-12 genome was reduced by making a series of planned, precise deletions. The multiple-deletion series (MDS™) strains (1), with genome reduction of up to 15%, were designed by identifying non-essential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving robust growth and protein production. Genome reduction also led to unanticipated beneficial properties, including high electroporation efficiency and accurate propagation of recombinant genes and plasmids that are unstable in other strains. Subsequent deletions and introduction of useful alleles produce strains suitable for many molecular biology applications. Recently, Scarab has built on the MDS™42 foundation strain, by creating the MDS™42 LowMut strain. It improves the already low mutation rate of the MDS™42 strain. The MDS™42 LowMut strain has been engineered to greatly reduce error-prone repair, which reduces the mutation rate to almost zero, even under the most stressful conditions, thus ensuring the most accurate replication of your plasmid.

Figure 1. MDS™42 LowMut has the Lowest Mutation Rate Under Stress. Mutation rates of various strains under unstressed and stressful conditions were determined. Stress conditions include overproduction of GFP, overproduction of a toxic peptide from pSG-ORF238 and treatment with mitomycin-C. All measurements were made using the cycA fluctuation assay, error bars represent 95% confidence intervals for the average of 3 independent measurements. BL21(DE3) failed to grow in the presence of 0.1 μg/ml mitomycin-C. ANOVA revealed a significance of p < 0.0001. Pairwise t-tests were conducted for each strain under a given condition compared to the corresponding MDS™42_lowmut strain. Figure 2: Non-Expressing Plasmid Mutations Accumulate rapidly in BL21(DE3), When a Toxic Methyltransferase is Overproduced. SinI methyltransferase was expressed from pSin32. Plasmids were isolated at various intervals and screened (by transformation in McrBC+ and McrBC- hosts) for mutations resulting in loss of function of the enzyme. Error bars represent 95% confidence intervals for the average of 3 independent measurements of mutant plasmid ratios. ANOVA revealed a significance of p < 0.005. Pairwise t-tests of each MDS™42_lowmut_mcrBC sample were done with the corresponding MDS™42 mcrBC and BL21(DE3) mcrBC sample, respectively. Starting from 10 hours, all MDS™42_lowmut_mcrBC samples differed significantly from the MDS™42 mcrBC (p < 0.01) or BL21(DE3) mcrBC (p < 0.005) samples. Figure 3: Multiple Deletion Strains tolerate "deleterious” genes. A chimeric gene composed of VP60 of rabbit hemorrhagic disease virus fused to the B subunit of cholera toxin (CTX) was very unstable in E. coli. Individually, both genes were stable in E. coli HB101, C600 and DH10B, but pCTXVP60 carrying the fusion gene in the same hosts did not produce fusion protein and was recovered in low yields. All recovered plasmids contained mutations in the CTXVP60 open reading frame, virtually all resulting from IS insertions. In contrast, the recombinant plasmid was completely stable in MDS™; normal yields of plasmid DNA were obtained. Representative restriction patterns of pCTXVP60. (A) Plasmid DNA from MDS™42 was transformed and propagated in the indicated host, then digested with NcoI and EcoRI. A representative of each restriction pattern was purified and sequenced. M, molecular weight marker, 1 kbp ladder; 1, MDS™41, no insertion; 2, MDS™42, no insertion; 3, DH10B, IS10 insertion; 4, DH10B, IS10 insertion/deletion; 5, C600, IS5 insertion; 6, C600, IS1 insertion; 7, C600, IS1 insertion. (B) Relative position of the IS element insertion sites in the CTXVP60 reading frame determined for the five examples presented. Figure 4: Plasmid stability in different host strains. Left: during four subcultures of pT-ITR, a plasmid with viral LTR segments; Lane 0, isolated plasmid DNA before subculture, lanes 1-4, successive subcultures. Plasmid DNA was digested with restriction enzymes and analyzed by agarose gel electrophoresis. KpnI cuts the plasmid at a single site, but in MG1655 two bands indicate a deletion in the plasmid. MscI cuts at two locations, but in MG1655 a third intermediate band confirms that the plasmid is deleted. Right: Stability of four variants of a Lentiviral expression plasmid in MDS™42 ΔrecA and Stbl3™ (Life Technologies), showing the proportion of transformants containing intact plasmids (Table 2 BioTechniques 43:466-470 (October 2007))(2).

Kit Components MDS™42 LowMut ΔrecA Electrocompetent Cells pUC19 Control DNA (10 pg/µl) SOC Medium Genotypes MG1655 multiple-deletion strain (1) ΔdinB ΔpolB ΔumuDC (2) ΔIS609 ΔpatD ΔydcV ΔydcU ΔydcT ΔydcS ΔydcR ΔhicA ΔhicB ΔyncJ ΔydcP ΔydcN ΔydcO ΔydcM ΔrecA(1819) The recA 1819 mutation is a deletion of recA. Quality Control Transformation efficiency is tested using pUC19 Control DNA, in duplicate. Transformed cells are plated onto LB plates containing 50 µg/ml carbenicillin. Transformation efficiency is > 5 x 10⁹ cfu/µg DNA. Storage Conditions Store components at –80°C. Do not store cells in liquid nitrogen.

White Glove IS Detection Kit

Product Manuals MDS™42 LowMut ΔrecA Electrocompetent Cell Kit Papers

1. Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6.
2. Csörgő et al. (2012) Low-Mutation-Rate, Reduced-Genome Escherichia coli an Improved Host for Faithful Maintenance of Engineered Genetic Constructs Microbial Cell Factories, 11:11.
3. Chacko S. Chakiath, CS & Esposito, D (2007): Improved recombinational stability of lentiviral expression vectors using reduced-genome Escherichia coli. BioTechniques 43:466-470.

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