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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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


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