Team:RPI Troy NY/Description
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Project Description
♦Background♦♦Design♦In this project, we sought to increase the production of sophorolipids by taking a two-part approach. In the first part, we engineered primers and inserted these into the yeast to be able to produce either normal amounts of sophorolipids or lesser amounts of sophorolipids (gene knockout). In the second part, we extracted these sophorolipids and completed analysis on this. 
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Protocols
♦Microbial Genetics Protocols♦Our Microbial Genetics group used PCR amplification and fusion PCR to construct our sequences with the modified genes. PCR amplification was used with primers designed to facilitate fusion PCR after the regions had been amplified. Primers were designed using Snapgene software and ordered from IDT Technologies. Melting temperatures of primers allowed for appropriate annealing and extension of the sequences during both the PCR amplification and fusion PCR phases. Stepwise fusion PCR allowed us to assemble the DNA parts in the appropriate order. The modified sequences were followed by a selection marker (hygromycin B gene) among additional terminators to allow for growth in the presence of antibiotic. Agarose GelsAnalytical Gel • 1-2% Agarose • Ethidium Bromide (1uL/100mL) Purification Gel • .8% Agarose • Ethidium Bromide (1uL/100mL) PCR Amplification• 50ng Template DNA • 25uL Accuzyme or Hotstart Mix (1uL/100mL) • 1uL primer stock (10mmol forward and reverse primer) • Remainder MilliQ • 1uL pure DMSO (Optional, use if high GC content) 50uL total Fusion PCR • 100ng each PCR amplicon (equimolar value)
• 25uL Accuzyme or Hotstart Mix (1uL/100mL)
• Remainder MilliQ
50uL total Run 10 cycles of appropriate annealing/joining protocol in Thermalcycler immediately add 1uL primer stock (10mmol forward and reverse primer) Run 20 cycles of appropriate annealing amplification and extension protocol in Thermalcycler BioRad Gel Extraction Kit Gels purified on .8% agarose gels Following steps might vary depending on number of samples. Dissolve ingredients in Distilled Water
Glucose (dextrose) should NOT be added to the broths before autoclaving; glucose should be made separately (500 g/L), with a final pH of 3.5 will be added in proportion to the nutrient broths (see above for final concentration of ingredients) to the culturing/fermentation flasks.
Adjust pH of both PM2 and FM2 broth to 4.5
Autoclave at 121*C for 15 minutes (setting 4)
Combine FM2 and PM2 and shake for about 48 Hours, then add Fatty acid (2x Feed) every 24 Hours and shake for 72. Collect samples and store in -20˚C Set up shake flasks, final volume should be 50 ml, with 1% PM2 and 1% of Fatty Acid This is the protocol we used to extract the sophorolipids: |
Experiments
Notebook
