The IS Detection kit is designed to test for the presence of transposable Insertion Sequences (IS) in a DNA of interest. IS elements are naturally present in the genomes of E. coli strains commonly used for protein and plasmid production. IS element transposition is known to be stimulated by the cell stress response and can lead to IS element “hopping” into plasmid DNA and or into other regions of the chromosome. Factors such the production of foreign proteins or the burden of carrying a high copy plasmid can induce the cell stress response. To alleviate these undesired transposition events, Scarab Genomics produced the Clean Genome® E. coli strains. These strains are devoid of all known IS elements (1-3) thereby creating the ideal hosts for the production of foreign proteins or plasmid DNA. This kit can be used to detect for the presence of all the specific known IS elements in the genomes of commonly used E. coli strains (Figure 1). It can also be used to determine which elements may have transposed into a plasmid grown in these strains. The kit also detects the presence or absence of known recombination hot spots (Rhs) in the E. coli genome.

Figure 1: IS Elements in popular E. coli strains. Each box shows the number of copies of the element in the genome. Note: these counts represent a snapshot in time. Strains that have been sub-cultured multiple times may differ in their IS count or contain different complements of IS elements. *Subsequent to the commercialization of the Clean Genome® E. coli strains, 2 copies of an atypical IS element named IS609 were recognized in the E. coli O157:H7 genome sequence (4). This IS element has not been shown to transpose, although other members of this IS family have been shown to transpose. The ability to transpose requires an intact orfA. The single IS609 element found in E. coli K-12 and B strains, however, carries a defective orfA with a stop codon mutation located near the middle of the ORF. IS609 has been removed in derivatives of the original MDS™ strains, indicated as “MDS™42 ΔMD64”. Figure 2. Detection of IS Elements in Plasmid Obtained from Commercial Sources. Detection of IS contamination in a commercial plasmid preparation of pBR322. Inward primers (panels a-d) or outward primers (panels e-h) specific for IS1, IS2, IS3, IS5, IS10, and IS186 were used (lanes 1-6, respectively; M is 1 kb+ size standard). Panels a and e show negative controls (no DNA), while positive controls in panels b and f are the individual IS elements cloned into pBR322. Panels c and g show purchased pBR322 and panels d and h show pBR322 isolated from MDS™42. PCR amplimers generated with outward primers specific for IS1, IS2, IS3, IS5, IS10 and IS186 were ligated, cloned with selection for tetracycline or ampicillin resistance, and sequenced (data not shown). Sequencing confirmed transposition of IS1, IS2, IS5, and IS10 to pBR322 in the commercial preparation. Figure 3. Workflow for pDNA production in Clean Genome® and unreduced E. coli strains. Extra care in the first steps will ensure trouble-free production. A) B) Figure 4: IS Primer Validation Using Water in Place of Sample DNA and Positive and Negative Control Genomic DNA. White Glove Kit protocol was followed using water in place of sample DNA. Panel (A) - Six microliters (6 μl) of the PCR amplification product was analyzed on 1.0% 1X TAE agarose gel. No products are visible when water is added in place of template DNA or when using the negative control genomic DNA. Positive control genomic DNA amplify as expected. Panel (B) - Lists the expected size of PCR product to be obtained using the positive and negative control genomic DNA.

Kit Components

• Positive Control Genomic DNA: 170 μl, sufficient for the analysis of 10 samples.
• Negative Control Genomic DNA: 170 μl, sufficient for the analysis of 10 samples.
• IS-specific Forward (F) and Reverse (R) Primers: 80 μl of each primer at a concentration of 5μM, sufficient for the analysis of 10 samples.

Forward Primers	Reverse Primer
IS1 Forward Primer	IS1 Reverse Primer
IS2 Forward Primer	IS2 Reverse Primer
IS3/ISEc17 Forward Primer	IS3/ISEc17 Reverse Primer
IS4 Forward Primer	IS4 Reverse Primer
IS5 Forward Primer	IS5 Reverse Primer
IS10 Forward Primer	IS10 Reverse Primer
IS30D Forward Primer	IS30D Reverse Primer
IS150 Forward Primer	IS150 Reverse Primer
IS186 Forward Primer	IS186 Reverse Primer
IS600/ISsd1 Forward Primer	IS600/ISsd1 Reverse Primer
IS609 Forward Primer	IS609 Reverse Primer
IS911 Forward Primer	IS911 Reverse Primer
ISEc1/3/5 Forward Primer	ISEc1/3/5 Reverse Primer
ISEc4 Forward Primer	ISEc4 Reverse Primer
RhsA/B/C Forward Primer	RhsA/B/C Reverse Primer
RhsD/E Forward Primer	RhsD/E Reverse Primer

• Positive Control dnaE Forward Primer and Positive Control dnaE Reverse Primers: 60 μl of each primer at a concentration of 5 μM, sufficient for the analysis of 10 samples.

MDS™42 Chemically Competent Cell Kit MDS™42 ΔrecA Chemically Competent Cell Kit MDS™42 ΔrecA Blue Chemically Competent Cell Kit MDS™42 Combination Package Chemically Competent Cell Kit ScarabXpress® T7 lac Chemically Competent Cell Kit MDS™42 Electrocompetent Cell Kit MDS™42 ΔrecA Electrocompetent Cell Kit MDS™42 ΔrecA Blue Electrocompetent Cell Kit MDS™42 ΔrecA trfA Electrocompetent Cell Kit MDS™42 ΔrecA trfA Blue Electrocompetent Cell Kit MDS™42 Combination Package Electrocompetent Cell Kit

Product Manuals White Glove IS Detection Kit Reports Production of DNA Vaccines Free from Mobile DNA Papers

1. Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6.
2. Kolisnychenko, V., Plunkett III, G., Herring, C.D., Fehér, T., Pósfai, J., Blattner, F.R., and Pósfai, G. Engineering a reduced Escherichia coli genome. Genome Research 12, 640-647 (2002).
3. Sharma, S.S., Blattner, F.R., and Harcum, S.W. Recombinant protein production in an Escherichia coli reduced genome strain. Metabolic Engineering 9, 133-141 (2007).
4. Perna, N.T., et al., 2001. Genome sequence of enterohemorrhagic Escherichia coli O157:H7. Nature 409: 529-533 (2001).

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