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™) platform exemplifies the “Clean Genome®” concept, providing bacterial strains with ideal characteristics for regulated biopharmaceutical applications:

• Scarab Genomics has engineered its Clean Genome® E. coli hosts for robust growth in minimal salts media, allowing production under strictly defined conditions.
• Plasmid and genomic stability are enhanced because transposable insertion sequences (I.S. elements) have been eliminated.
• Cultures are more stable because cryptic prophage were deleted, eliminating spontaneous cell lysis.
• Genes for toxins, virulence factors, flagella and fimbrae have also been removed to improve product purity and safety.
• Cells can continue to grow during protein expression because over 700 non-essential genes are eliminated and no longer compete for cellular resources, increasing metabolic efficiency
• High yields of recombinant protein drives down post fermentation processing costs.

The ScarabXpress T7 lac host strain carries the gene for T7 RNA polymerase on its chromosome under the control of a modified lac promoter and operator and is designed for use with T7 promoter based expression vectors. The dynamics of recombinant protein induction are significantly different in this host than in those experienced with the commonly used expression host BL21(DE3). In minimal medium lacking lactose, the ScarabXpress® T7 lac strain has more tightly regulated protein expression than BL21(DE3), providing the ability to reproducibly and precisely control induction. ScarabXpress T7 lac utilizes the wild-type lac promoter, which is of lower strength relative to the lacUV5 variant present in BL21(DE3). Moreover, the wild-type lac promoter is subject to regulation by the catabolite activator protein, CAP, whereas BL21(DE3) lacUV5 activity is largely CAP-independent due to a change in the CAP binding site. The properties of the wild-type promoter render it more sensitive to the effects of catabolite repression and promoter activation occurs gradually post-induction. This is in contrast to the rather abrupt expression of lacUV5 upon induction that is a reflection of its strength and CAP-independence.

The ScarabXpress® T7 lac strain is also more sensitive to repression; the promoter is more tightly regulated by LacI due to an alteration in a lac operator. The net result is a lowering of the background levels of expression relative to BL21(DE3). As a consequence of this tighter regulation, the ScarabXpress T7 lac host often works optimally with expression vectors that do NOT supply extra lac repressor from a plasmid encoded copy of the lacI gene. In multiple cases, significantly higher expression yields of a target protein have been observed by using a vintage pET vector i.e. one that carries neither the lacI gene nor a lac operator on its backbone. lacI-based pET plasmids were created to address the inherent leakiness of BL21(DE3). The additional lac repressor generated from this type of vector prevents full induction of the ScarabXpress® T7 lac host and may only yield optimal results when attempting to express proteins that substantially inhibit the growth of the Scarab host strain.

Figure 1. Inducer titration of pET9-TEST- PRO expression in BL21(DE3) and ScarabXpress®. Shake flask cultures were grown in 50 ml Korz minimal medium plus 0.2% glucose at 37°C to an OD600 of 0.01 then induced overnight with the indicated concentrations of IPTG. Cell pellets from each sample were obtained by centrifugation and soluble proteins were resuspended in BugBuster® protein extraction reagent for SDS-PAGE analsysis, loading the same OD600 equivalent in each lane. (M), molecular mass marker (sizes in kd at left edge of gel); arrowheads indicate TEST-PRO protein; chart at bottom indicates cell density (OD600) at harvest Figure 2: ScarabXpress®-1(T7 lac) yields 12X more protein than BL21. 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.

Kit Components ScarabXpress® T7 lac Chemically Competent Cells pUC19 Control DNA (10 pg/µl) SOC Medium Genotypes MDS™42 multiple-deletion strain (1) with a chromosomal copy of the T7 RNA Polymerase gene. 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.

White Glove IS Detection Kit

Product Manuals ScarabXpress® T7 lac Chemically Competent Cell Kit

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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