840:153g:Projects/project4/2009/04/30

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Project Summary

Summary of work completed and attempted

  • Extracted BioBrick parts from the registry of parts
  • Obtained Bacillus subtilis strains Globiformis and Niger from Dr. Walter and made glycerol stocks
  • Designed primers for aprE (alkaline protease subtilisin) gene using genomic sequence of B. subtilis strain 168 on NCBI
  • Extracted DNA from both strains but failed, then found a new protocol and got much better results
  • Attempted to clone the aprE gene using G & N strains but failed multiple times
  • Abandoned strains Globiformis and Niger due to possible genomic differences with strain 168
  • Found strain 168 online and ordered it from Ohio State University
  • Revived the new strain and made glycerol stocks
  • Extracted DNA from strain 168 using protocol from www.Bio.net
  • Used newly obtained DNA to clone aprE (alkaline protease subtilisin) and nprE (neutral protease bacillolysin) genes
  • nprE cloning failed even with multiple primers
  • Side project 1 abandoned!
  • Another side project was undertaken using the wintergreen gene
  • Using the wintergreen gene from last semester we tested the arabinose induced promoter in our plasmid of choice
  • Digestion failed and gene was never integrated into plasmid
  • Side project 2 abandoned!
  • Went back to original aprE project now that we got the new strain of Bacillus
  • Redesigned aprE primers by performing several mutations
  • Cloning succeeded using newly designed primers and DNA from strain 168!!
  • Extracted the PCR product from agarose gel and digested for ligation
  • Performed ligation and transformation into competent E. coli
  • Plated E. coli on amplicillin plates to select for transformed colonies
  • Extracted plasmid DNA from several colonies to verify presence of plasmid and aprE gene
  • Found just the plasmid, aprE gene was not present