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 Meta ΔrecA strain. It improves the already high density fermentation of the MDS™42 strain. The MDS™42 Meta ΔrecA strain’s optimized metabolism permits fermentation ≥OD300 in minimal media in ~24 hrs without glucose spike or cell lysis resulting in >40 g/L of a test protein at the 10 liter scale. It produced ~700 mg/L of pGWIZ GFP test plasmid in minimal media at the 10 liter scale without a temperature shift.

Figure 1. High Plasmid Yield without a Temperature Shift using Minimal Media. The MDS™42 Meta ΔrecA strain produced ~700 mg/L of pGWIZ GFP test plasmid in 10 liter scale fed batch fermentation without a temperature shift using Scarab’s ultra minimal media. Figure 2: 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 3: 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 Meta ΔrecA Electrocompetent Cell Kit pUC19 Control DNA (10 pg/µl) SOC Medium Genotypes MG1655 multiple-deletion strain (1) relA* Δrph ΔarpA ΔiclR ilvG⁺ ΔrecA(1819). 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 Meta ΔrecA Electrocompetent Cell Kit Papers

1. Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6.
2. 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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