Researchers generating biological materials often need to validate the identity of their production host strains for Quality Control and regulatory purposes. The Scarab Strain Identification (SSI) kit is designed to validate and verify the identity of Scarab Genomics’ Multiple Deletion Series (MDS™) Clean Genome® E. coli host strains. The MDS™ strains have been engineered by deleting over 15 % of the genome from the K12 reference strain MG1655, resulting in improved performance in many applications including production of biopharmaceuticals. As a side effect of the numerous genomic deletions, MDS™ host strains have missing or altered genes that affect secondary metabolic characteristics (e.g. nutritional requirements) that are sometimes used in traditional microbial identification. As a result, Clean Genome® strains are not always properly identified as “E. coli” when evaluated by growth phenotype tests. One set of tests using the BiOLOG Phenotype Microarray™ metabolic panel analysis identified MDS™42 as having a 95% probability of being a Citrobacter species rather than E. coli. This kit addresses the issues that may occur with phenotype based assays by providing a genome based approach wherein a panel of specific sequences unique to the MDS™ strains are assayed by PCR. The MDS™ strains are very distinct from other commonly used E. coli hosts due to the many genomic deletions generated during their creation. This kit uses primers designed to distinguish whether a given bacterial genomic DNA sample has or lacks a series of genomic modifications specific to the MDS™ strains.

A) B) Figure 1: Primer Validation with Positive and Negative Control Genomic DNAs and Citrobacter Genomic DNA and Expected Size Amplicon. (A) SDS-PAGE of PCR reaction products for the positive and negative controls. M = 1kb Plus DNA Ladder (Invitrogen) (B) Indicates the expected size of the resulting amplicon.

Kit Components

• Positive Control MDS™ Strain Genomic DNA: 160 μl, sufficient for the analysis of 10 samples.
• Positive Control ScarabXpress™ DNA: 50 μl, sufficient for the analysis of 10 samples.
• Negative Control K12 MG1655 E. coli Genomic DNA: 160 μl, sufficient for the analysis of 10 samples.
• Negative Control Citrobacter Genomic DNA: 160 μl, sufficient for the analysis of 10 samples.
• SSI-specific Forward (F) and Reverse (R) Primers: 80 μl of each primer at a concentration of 5 μM, sufficient for the analysis of 10 samples.

  Forward Primers	Reverse Primer
  F-SSI-Del A	R-SSI-Del A
  F-SSI-Del B	R-SSI-Del B
  F-SSI-Del C	R-SSI-Del C
  F-SSI-Del D	R-SSI-Del D
  F-SSI-Del E	R-SSI-Del E
  F-SSI-Del F1	R-SSI-Del F1
  F-SSI-Del F2	R-SSI-Del F2
  F-SSI-Del G1	R-SSI-Del G1
  F-SSI-Del G2	R-SSI-Del G2
  F-SSI-Del H1	R-SSI-Del h2
  F-SSI-Del I	R-SSI-Del I
  F-SSI-Del J	R-SSI-Del J
  F-SSI-Del K	R-SSI-Del K

• tufA SSI Positive Control Forward (F) and Reverse (R) Primers (SSI-Pos Cntrl): 60 μl of each primer at a concentration of 5 μM, sufficient for the analysis of 10 samples.

White Glove IS Detection Kit

Product Manuals Scarab Strain Identification Kit Papers

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

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