Team:Washington/Protocols

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Protocols

• Yeast Chromosome Integration
• Theophylline Stock
• Fluorescence Reading

Digest E. Coli plasmid using PmeI restriction enzyme

• 1 ug of DNA
• 5 uL of 10x NEB CutSmart buffer
• 1 uL of restriction enzyme
• Fill to 50 uL with water
• Incubate at 37 C for 15 minutes (1 hour if not using TimeSaver buffer)
• Heat inactivate at 65 C for 20 minutes
• Agarose gel purify (optional)

Salmon Sperm Transformation

• Grow a yeast overnight
• Check OD of culture. 0.5-0.6 are the preferred readings, if the reading is lower, wait for longer growth, if the reading is higher, dilute the sample.
• Spin down 10 ml of cells per transformation.
• Decant supernatant and wash with 10 ml ddH2O. Vortex to resuspend and spin down.
• Remove the supernatant.
• Resuspend cells in 300 uL .1 M LiOAc. Transfer to a 1.5 mL tube.
• Incubate at 30 C for 15 min
• Put salmon sperm DNA in boiling water for 5 minutes. Cool immediately on ice.
• Spin down cells and remove supernatant.
• Add the following in order:
1. 240 uL 50% PEG
2. 36 uL 1.0 M LioAc
3. 10 uL salmon sperm DNA
4. 34 uL DNA
5. 40 uL ddH2O
6. Final volume: 360 uL

The auxin pathway produces a color output rather than a fluorescent one….(I will finish this part tomorrow when my computer isn’t dead) Microfluidic design:.....(1% remaining….)

In this design RNA aptamers are used to sense our target molecule, theophylline. Aptamers, unlike antibodies, can actually bind to virtually any molecule, allowing for a more versatile system. We’ve implemented a ribozyme switch which, when active, cleaves the mRNA code of our target sequence, hindering the production of GFP by default. However, in the presence of theophylline our switch becomes inactive, allowing for the expression of our target gene. This system is useful because it is faster-acting than more traditional expression pathways, and can be generalized to many other small molecules by changing the aptamer sequence.

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For commercial shellfish farmers and recreational hunters alike, marine biotoxins pose a significant threat to health and welfare. With this project, we aim to create an inexpensive and easy-to-use test kit for the detection of the shellfish toxin okadaic acid using engineered yeast strains and DNA aptamers on a paper device. We also hope that this project paves the way for a new class of biosensors capable of detecting a wide range of small molecules.