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MDS™42 Meta Chemically Competent Cell Kit

: MDS™42 Meta Chemically Competent Cell Kit

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SKU Product Manufacturer Price: Quantity
C-6265-05 C-6265-05K - MDS™42 Meta Chemically Competent Cells ***** 1
C-6265-10 C-6265-10K - MDS™42 Meta Chemically Competent Cells Scarab ***** 1
C-6265-20 C-6265-20K - MDS™42 Meta Chemically Competent Cells Scarab ***** 1
  • Ultra High Density Fermentation Lead to Ultra Yields - MDS™42 Meta strain, improves the already high density fermentation of the MDS™42 strain. The MDS™42 Meta strain optimized metabolism permits fermentation ~OD300 using Scarab’s ultra 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. The MDS™42 Meta strain produced ~700 mg/L of pGWIZ GFP test plasmid in minimal media at the 10 liter scale without a temperature shift.
  • IS-free Plasmid Production - Recombinogenic or mobile DNA elements such as Insertion Elements have been deleted from the Clean Genome® E. coli strains to ensure faithful replication of your construct.
  • Maintain and propagate unstable clones and plasmids – pDNA vectors carrying direct repeats, inverted repeats or other sequences that result in pronounced secondary “stem-loop” structures are commonly unstable in standard E. coli hosts. Clean Genome® E. coli strains are frequently used for cloning sequences that suffer unwanted recombination events in other strains.
  • Clone and Express “deleterious” genes – Clean Genome® E. coli strains’ natural defenses such as IS elements that mutate “deleterious” genes to stop the stress on the organism have been deleted so they cannot interfere with your experiments.

Background

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 strain. It improves the already high density fermentation of the MDS™42 strain. The MDS™42 Meta 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.

Figures


Figure 1. High Plasmid Yield without a Temperature Shift using Minimal Media.
The MDS™42 Meta 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).

Specifications

Kit Components
MDS™42 Meta Chemically Competent Cells
pUC19 Control DNA (10 pg/µl)
SOC Medium

Genotypes
MG1655 multiple-deletion strain (1) relA* Δrph ΔarpA ΔiclR ilvG+.

Quality Control
Transformation efficiency is tested using pUC19 control DNA, performed in duplicate. Transformed cells are plated on LB plates containing 50 μg/ml carbenicillin. Transformation efficiency is ≥1x108 cfu/μg DNA.

Storage Conditions
Store components at –80°C. Do not store cells in liquid nitrogen.

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Patents & Disclaimers

Products are sold for non-commercial use only, under Scarab Genomics limited use label license: Limited Label Use.

Scarab is providing you with this Material subject to the non-transferable right to use the subject amount of the Material for your research at your academic institution. The Recipient agrees not to sell or otherwise transfer this Material, or anything derived or produced from the Material to a third party. NO RIGHTS ARE PROVIDED TO USE THE MATERIAL OR ANYTHING DERIVED OR PRODUCED FROM THE MATERIAL FOR COMMERCIAL PURPOSES. If the Recipient makes any changes to the chromosome of the Material that results in an invention in breach of this limited license, then Scarab will have a worldwide, exclusive, royalty-free license to such invention whether patentable or not. If the Recipient is not willing to accept the terms of this limited license, Scarab is willing to accept return of this product with a full refund, minus shipping and handling costs. For information on obtaining a license to this Material for purposes other than research, please contact Scarab’s Licensing Department. Scarab Genomics’ technology is covered by U.S. Pat. No. 6,989,265 and related foreign applications.

Clean Genome® is a registered trademark of Scarab Genomics, LLC.