fishPHARM

A novel drug delivery system and probiotic treatment for Bacterial Coldwater Disease and app for monitoring nutrient content in fish hatcheries.  iGEM 2015.

Below is an overview of the project.

THe Problem

Bacterial coldwater disease (BCWD) is the single most devastating fish disease on the planet and in our fish farms. Caused by a strain of bacteria called Flavobacterium psychrophilum, this single infection has been found to kill 1 in every 4 fish it infects.  Because of the ease of transmission, bacterial coldwater disease is especially prevalent in industrial aquaculture and fish farming where fish live in dense populations. Outbreaks are common, observed globally, and result in harsh economic effects on commercial salmonid producers and fisheries.The more that we depend on aquaculture, the more easily catastrophic diseases like bacterial coldwater disease can endanger our environment, our economic welfare, and even our own well-being.  To solve this problem, Cornell iGEM presents to you fishPHARM.

Our goal is to rid the world of an agonizing fish killer and pave the way for a novel new treatment for bacterial diseases.

Upon infecting the fish, F. psychrophilum gradually eats away at the flesh, leaving horrible skin lesions. In later stages, the disease causes spinal cord deformities, giving its host a slow and painful death. To add salt to the wound, this disease can be transmitted both horizontally and vertically.  In 2004, a case study was done by the Department of Ecology at Montana State University to analyze the effects of Bacterial Coldwater Disease at Washoe Park State Fish Hatchery.  Washoe Park typically loses 30 to 45% of its westslope cutthroat trout production to the disease annually and currently BCWD is the only fish disease found in state fish hatcheries in Montana.

Currently, no economic and consumer-safe industrial treatment exists for bacterial coldwater disease.  Antibiotics, such as terramycin, are placed directly in the water, where they leach into the surrounding environments and invade other ecosystems and marine life.  Furthermore, the use of antibiotics increases the flavobacteria’s resistance until it is no longer even an operative means of treatment.

MY Role

I was a Wetlab Engineer and the expert in our wetlab endeavors to create our treatment.  I presented with two of my teammates at the iGEM World Jamboree Competition at the Heinz Convention Center in September 2015 against 280 other international university teams.  I also designed and coded our project wiki (see link above), and created animations of our fishbit drug delivery system for our wiki and presentation.  We won a Gold Medal and three category awards, one of only two teams in North America to win more than two category awards that year- Best Environmental Project, Best Supporting Entrepreneurship, and Best Applied Design!

OUr Solution

fishPHARM is comprised of these four key components:

1) Flavocide, a biologically engineered ecnB peptide suspended in a biodegradable copolymer, a small protein that is toxic to the Flavobacterium, yet completely safe for the fish and for human consumption.

2) The fishbit, our novel, easy-to- use drug delivery system that integrates the ecnB peptide and allows for the time-dependent release of our treatment into the fish.

3) Our Heimdall fish app, a preventative monitoring system fish farmers can use in combination with the fishBIT to keep their hatcheries BCWD-free.

4) We worked in collaboration with local upstate New York fish hatcheries to improve and redesign our novel bacterial coldwater disease treatment system to better fit the needs of our product market, and have created a business model to further the global scope of our fishPHARM’s impact.

Flavocide

Flavocide is our biodegradable peptide to cure Bacterial coldwater disease.

Flavobacterium psychrophilum is a gram-negative, aerobic, bacterium that is ubiquitous to freshwater.  Since its initial discovery in 1948, F. psychrophilum has been isolated from many salmonid-producing regions of the world. Flavocide is composed of a toxic peptide called entericidin B or ecnB for short.  

Antidote/toxin gene pairs known as "addiction modules" can maintain plasmids in bacterial populations through post-segregational killing. The entericidin locus encodes tandem paralogous genes ecnA and ecnB, and directs the synthesis of the small cell-envelope lipoproteins.The entericidin A lipoprotein is an antidote to the bacteriolytic entericidin B lipoprotein.  EcnB is a typically 48 amino-acid sequence located on the inner membrane and is responsible for the probiotic activity of Enterobacter C6-6, an autochthonous strain that inhibits the in vitro growth of F. psychrophilum, and when ingested as a probiotic, provides protection against coldwater disease in rainbow trout.

We have engineered BL21 E. coli for the production of ecnB.  Hundreds of bacterial entericidin B phenotypes exist naturally, and we wanted to find the most effective one at killing Flavobacterium to develop flavocide.  Of all these different isoforms, we narrowed it down to 20 that most comprehensively represented all isoforms based on their phylogenetic relationships. 

Testing

We confirmed the expression of our BioBricks using Western blots, and then conducted a lysate assay, and the initial results were very promising for the MBP+EcnB fusion protein construct. We added liquid cell lysate from each of our biobricks to liquid cultures of flavo, and monitored the growth of the bacteria over time. We further validated our constructs with a series of ZOI assays in which disks soaked with lysate from our ecnB biobricks were placed on plates of flavo. Our controls all displayed no zones of inhibition at all in the presence of F. psychrophilum (as expected), but the lysates of three EcnB constructs all showed very consistent fields of inhibition. From this, we can conclude that these three tested EcnB constructs all are effective in combating F. psychrophilum growth.

We PResent a cure

In fact, our MBP EcnB construct from M. haemolytica performed especially well in inhibiting the growth of flavo. This construct generated an average zone of inhibition almost identical in diameter to the average zone of inhibition from oxytetracycline, an antibiotic currently used in fish farms to treat Bacterial Coldwater Disease.  Our construct and flavocide is comparable to antibiotics currently used in combatting BCWD. Flavocide is our cure.

fishbit

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.

fishBIT is 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.

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.

Heimdall Monitoring System

Named after the Norse god of surveillance, the Heimdall Monitoring System 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.

The HMS will be prototyped extensively to develop a more efficient design, and its mobile app will continue to be streamlined.