Gene Disrupted Strains - About

About

   On April 1st 2008, it started to distribute the gene disrupted strains of Bacillus subtilis. This gene disrupted strains library (a wild type and 2514 gene disrupted strains) is based on the strains newly reported in Kobayashi et al.(2003 PNAS vol.100 4678-4683). In this project we checked all strains by PCR about whether these strains were correctly disrupted or not and started to distribute the confirmed strains.
   PCR experiments for 2514 strains were done as following. As first inspection, two parts of each genome were checked for all strains. One part was the region between initiation codon of the putative disrupted gene and pMUTIN vector sequence(1). The other part was the region between termination codon of the putative disrupted gene and pMUTIN vector sequence(2). Then the strains were confirmed as gene disrupted strains, in which an expected DNA fragment was amplified. Next, as second inspection, the region between initiation codon and termination codon of the putative disrupted gene was checked for the strains in which neither PCR was amplified in the first inspection(3). Then the strains were also confirmed as gene disrupted strains, in which an estimated 10kb DNA fragment including the disrupted gene and pMUTIN vector sequence was amplified.
   From above two inspections, a wild type strain and 2090 gene disrupted strains are available for the distribution. (Keita Aoki)
   During cultivation of the gene disrupted mutants, erythromycin should be added to culture at final concentrations of 0.5 ug/ml to prevent pMUTIN from popping-out. Although rate of popping-out may depend on individual mutants, the rate in strain MGNA-B928, for example, is about 25%.
   Quality verification of gene disrupted strains was largely performed. As a result, 243 strains have passed the test for distribution. 2,335 strains including a wild-type strain are now ready to be distributed (April, 2016).

Method of gene disruption

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Method of quality check

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