Team:Washington/Aptamer

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Aptamer Background

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.

An aptamer is a single strand of RNA which folds into a structure that is able to bind to a variety of small molecules and proteins. Theophylline is commonly used target molecule for academic studies on aptamers due to its ability to permeate membranes.

Ribozymes are self-cleaving pieces of RNA. This can be utilized to destabilize RNA transcripts.

Aptazymes are a combination of both aptamers and ribozymes. This allows for the selectivity of aptamers which in turn results in the activity of the ribozyme section of the RNA. Overall this is a reactive strand of RNA with selectivity. Additionally, this can control protein expression at the level of translation. This allows for quicker response times. RNA allows for easy tenability so that the general system can be modified to fit several contexts. In this project, we use theophiline as a model small molecule due to its ability to permeate through membranes.

Aptazyme 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:

240 uL 50% PEG

36 uL 1.0 M LioAc

10 uL salmon sperm DNA

34 uL DNA

40 uL ddH2O

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.

Design