Cornell iGEM

Dry Lab Overview

fishPHARM’s mechanical engineering work centers around two main principles: 1) the real-world implementation of a peptide treatment for bacterial coldwater disease (BCWD), and 2) the prevention of bacterial coldwater disease outbreaks on fish farms. To address these goals, fishPHARM has developed engineering solutions for the individualized delivery of flavocide, our bioengineered peptide treatment for BCWD, as well as a fish farm tank monitoring system for the constant monitoring of aquaculture conditions.

fishPHARM presents fishBIT, a novel means of delivering time-released flavocide gel to each fish individually. Inspired by fish tags, devices used to track fish in the wild, fishBIT allows fish farmers to target specific fish for flavocide treatment. Farmers can choose to apply the fishBIT system to a few affected fish, or to their whole farms. Each delivery device can store enough flavocide to treat one fish for the prescribed dosing period, as well as be easily re-dosed, making it an effective means of treating multiple and recurring infections. The device operates through passive diffusion of flavocide suspended in a medical hydrogel. FishBIT is safe for the fish, and is constructed using materials which are all approved by the FDA. Testing of the fishBIT system in a hydraulic flow channel has shown that fishBIT can resist normal fish motion and speeds.

FishPHARM presents the Heimdall Monitoring System (HMS). Named after the Norse god of surveillance, the HMS consists of sensor hardware to be installed within aquaculture tanks, as well as a mobile app for the continuous monitoring and collection of temperature, flow rate, dissolved oxygen concentration, and nitrate concentration. These factors are important in the health of a fish hatchery, and if improperly maintained, will lead to infections and higher mortality rates. Current methods of monitoring these factors are inefficient and not as reactive to changes in conditions. The HMS is still in its development phase. A first prototype of the hardware is in the process of being built, and the mobile app is ready for testing.

Future work will be done at fishPHARM to improve the design and manufacturing of fishBIT for mass production. Additional testing will be conducted as we plan to apply for FDA approval of the device. The HMS will be prototyped extensively to develop a more efficient design, and its mobile app will continue to be streamlined.

Components

The fishBIT system combines the simplicity of standard fish tags with the novelty of a synthetic biology treatment.

The plastic harpoon region was designed to ensure that the tag remains in its host under most forces. Its center was hollowed out to allow contact between the host interior and our synthetic biology treatment. Moreover, this plastic is an FDA-approved biocompatible material to minimize risks to the host.

The silicone-based biocompatible tubing houses the treatment gel. It also serves as a marker to determine how much gel has dissolved.

The bright red gel consists of the entericidin protein contained in FDA-approved poly(lactic-co-glycolic acid). This copolymer dissolves readily in most fluids, and its properties can be easily altered. Furthermore, both the protein and the gel are both environmentally safe; the peptide is ubiquitous and the gel degrades easily.

Our tag applicator was designed to be used just like conventional applicators, but with added comfort and easier handling. fishPHARM is currently working on an alternative, more efficient applicator that is inspired by firearm trigger mechanisms.

Each fishBIT starter kits comes with 20 fishBIT tags, a tag applicator, a vial containing our novel flavocide, a needle, a syringe, and an information booklet.

See animations of our components here.

Fabrication & Assembly

The fish tag was 3D printed instead of manually fabricated for increased prototype precision. The Freeform Pico 3D printer from Asiga was used to print the fish tag for its high resolution printing abilities. The fish tag applicator, however, was machined in Cornell's Emerson Lab machine shop out of aluminum. A tube with an inner diameter of 1/16" and outer diameter of 1/8" was attached to the back of the fish tag to hold the hydrogel. A syringe was used to insert the hydrogel into the tube, and a clamp was placed at the end to prevent hydrogel escape. The fish tags and applicators, along with the peptide hydrogel, was packaged into a starter kit for distribution to fish farmers.

Testing

We at fishPHARM believe in safety and responsibility for fish above all. Therefore, we have put the fishBIT system through a rigorous testing process to ensure maximum durability and hydrodynamic properties and minimal invasiveness. We have simulated in the lab our fishBIT prototype working under the real world conditions that the device will encounter in an aquatic ecosystem. In doing so, we have proven its effectiveness as well as optimize its design. Additionally, we are constantly fine-tuning the tag and applicator design based off of implementation and feedback from our fish hatchery collaborators.

Ballistics Testing
We wanted to ensure the fishBIT system was less invasive than current methods of tagging fish. We injected fishBIT and standard fish tags (from fishtagger.com) into a 1% concentration clear agar block to qualitatively assess damage done to fish tissue. FishBIT and the standard tags were applied with their respective applicators. The pictures below represent our first iteration through the design process. Our applicator was able to make a clean puncture and firmly place the fish tag within the agar. Additionally, our fish tag was able to successfully remain inside the agar (more testing about the effect of water flow on fish tag embedding is discussed in the “Hydrodynamics” section). However, as seen by the effect on the agar, both our applicator and tag should be made narrower in order to inflict less trauma on the fish tissue. The conclusions of our ballistics testing are also supported by the feedback from the Rome and Bath fish hatcheries.

We also wanted to make sure our applicator was as effective as the standard applicator in penetrating the skin and scales of salmon. Therefore, we used a diseased salmon and injected both systems into the tissue. Based on our testing on the agar block, we expected fishBIT to not be as effective as the standard tag. However, our applicator was just as effective as the control applicator in penetrating the scales of the fish, and did not cause significantly more damage to the fish tissue. The control tag and the fishBIT tag both resisted a gentle tug on the exposed portion. Tugging gently on fish tags after applying them is standard procedure for tagging fish, and is a good test for the tenacity of the newly placed tag [1]. These figures demonstrate the effectiveness of fishBIT relative to that of a standard tag (in yellow).

Hydrodynamics
We performed hydrodynamics testing in Cornell's DeFrees Hydraulics Lab. We tested fishBIT at the maximum speed of the Tilting Wind-Water Tunnel, which is approximately 1 m/s. While the maximum burst speed of salmon is 4.13 m/s, this only occurs in nature when salmon swim upstream in the mating process. This maximum speed is never observed in salmon in net pens because the tight living conditions prevent salmon from reaching very high speeds. Thus testing fishBIT at 1 m/s is indicative of the conditions that salmon using fishBIT will be living in.
The main purpose of hydrodynamics testing was to see how well water at high speeds is able to flow over the fish tag. Thus, an orange was used in place of salmon tissue because the purpose of the testing was mainly to observe the flow of water over the part of the tag that is exposed outside the fish’s body. FishBIT was tested in different water conditions by positioning two oranges at varying heights inside the Tilting Wind-Water Tunnel. The orange near the surface of the water represented turbulent water conditions that fishBIT would have to withstand, and the orange submerged approximately 10 cm under the surface of the water represented faster flowing water conditions. Overall fishBIT was able to handle both conditions very well - the clamp stayed in place, and the tag did not twist in a manner that would impeded the natural movements of the fish. One of the improvements that drylab is considering for the future is using a smaller clamp on our fish tag in order to have the tag maintain smooth movements in turbulent waters.

Future Testing

Stress testing - We plan on using a stress machine to apply loads to the fishBIT barb, head/tubing interface, and clamp/tubing interface, in order to find maximum load capabilities. These would be the forces under which we would expect to see any failure of the system.

Tensile strength - Cornell University offers an Instron 8500 Load Frame and data Acquisition System as well as multiple strain gauges. This would allow the estimation of the tensile strength of fishBIT, and to readjust the design or incorporate more durable materials if necessary. Alternatively, a simpler test would be to attach weights to the fishBIT. This would also provide a reasonable estimate of the tensile strength.

Safety

Animal and environmental safety guided the fishBIT and flavocide hydrogel design and development. Industry standards for salmon were used to develop the size and shape of the fish tag [2]. Cornell iGEM’s main goal was creating an efficient and localized drug delivery system that, unlike with current antibiotic treatments, does not have significant drug escape into the environment. FDA-approved, biocompatible, and environmentally safe PLGA polymer will used to create the peptide hydrogel [3].

Future Work

FishBIT is a novel solution to drug delivery in fish. While it is a great method of delivering flavocide to sick fish, we at fishPHARM believe in always challenging ourselves to find new and better solutions. While working on a formula and dosing for Flavocide is our number one priority, we will also be exploring new ways of delivering the time-released medication to affected fish. One possible future method of delivering Flavocide would be a pneumatic injection system, which injects individual capsules in fish for rapid and accurate delivery. This system would make treating fish a much less time-consuming process, and would increase the appeal of the fishPHARM BCWD treatment method.

References

[1]How to: Tagging Instructions. (n.d.). Retrieved September 18, 2015, from https://www.taglouisiana.com/how_to_guide?came_from=what

[2] Fish Tag Types and Locations. (2015). Retrieved August 1, 2015, from http://www.dfg.ca.gov/fish/Fishing/Monitoring/FTag/FTag_Desc.asp

[3] Makadia, H., & Siegel, S. (2011, August 26). Poly Lactic-i-co-i-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Retrieved August 1, 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347861/