120 journal reporting
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120 journal reporting

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120 journal reporting 120 journal reporting Presentation Transcript

  • Comparative transcriptional analysis ofBacillus subtilis cells overproducingeither secreted proteins, lipoproteins ormembrane proteins
  • Cell Factory• Cells capable of secreting valuable proteins• GRAS (Generally Recognized as Safe)microorganism• Escherichia coli• Lactobacillus spp.
  • Problem• Which of the 8 secretory proteins of B. subtilisNZ8900 is/are found in large amounts in thegrowth medium?
  • Methodology1. 8 genes cloned using SURE systemoverexpression vector pNZ8902 or pNZ89012. Genes were fused to C-terminal 6His-tagencoding sequences for imunnodetection3. Bacterial cultures grown to midexponentialphase and gene expression was induced withsubtilin4. Testing protein production (whole cell extract;extracellular; membrane; cytoplasmic) andMicroarray analyses
  • Result and DiscussionSDS-Page Immunodetection
  • Result and DiscussionSDS-Page Immunodetection
  • Conclusion• Data reveal possible bottlenecks in theprotein overproduction process• Recombinations in the genes controlling theexpression of XynA and NprE would producevaluable proteins in high amounts fit forindustrial production
  • Microarray Analyses• measure the expression levels of largenumbers of genes simultaneously orto genotype multiple regions of a genome
  • General Effects of Overproduction• Downregulation of the sdpABC (sporulating delayprotein) operon– Role in apoptosis– Kills non-sporulating siblings and feeding on the deadcells under conditions of nutrient limitation• Upregulation of class I heat-shock genes codingfor molecular chaperon groES and groEL– Protects the cell from toxic accumulation of mis- orunfolded protein