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Degradation Notebook
June 22, 2012
Created a spreadsheet detailing various possible bacterial strains and plasmids to be used for complex polymer degradation
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June 25 - 29, 2012
Sent out requests for bacterial strains and plasmids based on spreadsheet
Emailed Dr. Jared Leadbetter asking if he has strains + protocol for lignocellulose plates
Made carb. R plates
Made minimal media agar plates, bottom layer of overlay plates
Met with Professor Leadbetter to discuss difficulties with creating enrichment plates and finding bacterial samples
Will be following up on some of his suggestions
Notes from Meeting with Professor Jared Leadbetter from June 28th
Overlay Plate Options
- Top layer with minimal media, although either minimal media or just water + agarose should work
- Embed bacteria into top layer mix
- Use filter paper made of chosen carbon sources
Bacterial Strains
- Ponds around campus
- Beach
- Teredinibacter turnerae gen: helps degrade wood in shipworms (http://ijs.sgmjournals.org/content/52/6/2261.abstract)
May also choose a different host organism for transformation
- Instead of using E. coli can try using yeast (simplifies problem to only of degradation, and not also ethanol synthesis)
- Can also try bacteria found by Aztecs to make alcohol (Zymomonas mobilis)
Emailed for samples of nitrocellulose, latex, and teflon membranes.
Researched Z. mobilis.
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July 2-6, 2012
Researched zymomonas mobilis, narrowed down focus to strain ZM4
Gathered more information on protocols specific to that strain.
Looked up additional information about potential vectors to use to transform z. mobilis
Requested p42-0119 plasmid for expression z. mobilis from the Oak Ridge National Laboratory
Made 20% glucose solution stock and RM plates for Z. mobilis
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July 9-13, 2012
Ordered strains from the ATCC.
Discussed Biolog plates idea
Research z mobilis degradation 5 and 6 carbon sugars
Researched for genetic sequences for sugar degradation enzymes.
Sent in request for oligos sequences for alpha-gluctosidase gene (can degrade maltose)
Made tetracycline plates
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July 16-20, 2012
Electroporated pSB1T3 into competent E coli cells, incubated in LB for an hour, and streaked transformants onto tetra plates
Streaked DH5alpha E. coli strain with plasmid pMQ30 onto a gentamicin 20 ug/mL plate
Streaked Z. mobilis ZM4 from ATCC onto a RM plate and liquid culture.
Streaked out DKN79 (DH5alpha + pLAFR5) onto a tetracycline plate
Streaked out DKN1005 (BW20427 [WM3064] + pLAFR5) onto a tetracycline plate
Made LB media
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