Environmental Sample Processor

1. Environmental Sample Processor Barbara Wright

2. Environmental Sample Processor • Chris Sholin of MBARI • 14 years of development • NASA grant of 3 million dollers • Detects microorganisms with DNA probes • Is able to do tests under water that were previously only done in a lab

3. DNA Probe Technology • Uses synthetic nucleic acid sequence probes that will bind to known target sequences from marine organisms • Binding of probe and target DNA causes change in fluorescence which can be recorded by a camera • Two types of DNA probes

4. Sandwich hybridization Assay • DNA probe + biotin (vitamin B7) + streptdavidin + filter support. • biotin/streptdavidin complex anchors the DNA probes to the assay plate. • target mRNA binds to the probes. • more DNA probes introduced which bind to a different region of the target mRNA. • This creates a “sandwich” of probe-target mRNA-probe. • The second probe is able to produce light that is then captured by the ESP’s camera. Picture taken by camera, light indicates presence of specific organisms

5. Enzyme-linked immunosorbent assay (ELISA) • Used with toxic bacteria • known toxic proteins are attached to the plate with an adhesion protein • the water sample is combined with a solution containing antibodies to the target toxin which are complexed with a light emitting protein. • If the target toxin is present in the water sample the introduced antibodies will bind to it and be removed with the sample • if there are no target toxins in the water sample the antibodies will bind to the toxins on the assay plate and will not be removed with the solution. • light from the antibodies will be captured by the camera. In ELISA, the absence of light indicates the presence of organisms. Sample toxins and antibodies present = no light Antibodies present, no sample toxins = light emitted

6. Pucks • Where all the action happens • Size of a quarter and 1/2 inch high • Houses filtration, mixing of reagents, DNA analysis, and storage • Water samples and reagents moved from puck to puck with syringes • Syringes can only hold 25 milliliters at a time so a 1 liter water sample can take an hour to process.

7. Pucks held by rotating carousal of cylinders Pucks raised in cylinder by a mechanical elevator At top they are lifted by a mechanical arm and placed into the processing station Reagents are then added to puck with syringes out of an IV bag Movement of Pucks

8. Allows ESP to sample water from deep in the ocean (4000m) External system that can be attached to ESP Uses pistons to change the pressure of water to 1 atm (usable by ESP) Deep Water Sampling Module The two pressure changing cylinders with pistons

9. Plans For ESP • Short time at MARS site in Monterey Bay • 1 year at NEPTUNE site at axial seamount • Active underwater volcano • Identify organisms at earth’s crust • Measure effects of seafloor flow (due to volcano) on microbial populations • Environment thought to be similar to planetary oceans Active volcano

10. Polymerase Chain Reaction MFB component of ESP - Microfluidic Block (MFB) holds reagents for reactions - Current research focused on modifying it for the PCR reaction - PCR amplyfies DNA sequences by making hundreds of copies - PCR requires enzymes, nucleic acid extracts and heating and cooling cycle - Current ESP has heating and cooling cycle for DNA extraction - PCR modification would include additional reagent bags, syringes, and new software

11. Europa • Current funding for ESP comes from the NASA program for exploration of life on other planets • Jupiter’s moon Europa is thought to be covered by an ice incased ocean • If the ESP could access Europa’s ocean, it could determine if there was life (microorganisms) in it.

12. Questions? Thank You!