Project Overview

Cornell University is situated in the heart of the Finger Lakes region of Upstate New York State where a diverse array of salmonid fish call home. Unfortunately, the region has also been impacted by a series of bacterial coldwater disease (BCWD) outbreaks, severely reducing the number of fish available for recreational fishing and local aquaculture. BCWD is a potentially lethal bacterial infection of salmonid fish species caused by the pathogen Flavobacterium psychrophilum. Fish suffering from BCWD develop skin lesions that effectively renders the fish inedible and unviable. The disease is not only found in Upstate New York, but also has been cited in other fish farms and hatcheries where fish are raised in close quarters. In fact, other American fish farms have reported instances in which over over 30-45% of trout raised have been lost due to BCWD (Ryce 2004). The grave economic and agricultural consequences of BCWD are unresolved by current means of treatment consisting of antibiotics. This year, Cornell iGEM has developed fishPHARM: a comprehensive prevention and treatment plan that uses the tools of synthetic biology to combat BCWD.

Since the etiology of BCWD is characterized by intra-organismal microbial proliferation our treatment protocol seeks to reduce microbial loads within the infected fish through the use of a peptide called Entericidin B. Recent studies have shown the peptide to be toxic to the growth of F. psychrophilum, thereby providing a potential treatment to BCWD[1]. Hundreds of bacterial entericidin phenotypes exist naturally. This year, Cornell iGEM has engineered over 20 strains of Escherichia coli for the regulated production of such peptides to test their efficacy against the growth of F. psychrophilum. In doing so, we plan to develop the most effective probiotic treatment for BCWD and advance the study of therapeutic probiotic treatments in combatting similar diseases.

In addition, the team plans to engineer a novel fish drug delivery system to safely administer our probiotic treatment without environmental harm. We have developed a working prototype of a fish tag that can safely latch onto the skin of a fish and demonstrate the secure and time-effective release of our peptide to treat BCWD in an infected fish. Our fish tag aligns with current practices of fish tagging in the aquaculture industry today, and has immediate potential to be implemented on both a local and global scale. We have shared the device with local hatcheries and fish farms to both garner feedback to improve prototype designs as well as help local farmers combat coldwater disease.

Success of this putative microbial control system holds important ramifications for human and animal health, as similar treatment methods could be devised for bacterial infections among humans. Prevention of BCWD will help provide a more secure, sustainable future in the fish industry and beyond to aid growing population necessary to sustain the continued increase of the human population.

Project Background

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Safety

Cornell iGEM understands the inherent risks of working in a lab facility and aims to take all necessary precautions to ensure no personal or environmental harm occurs. To this end, we have implemented the following safety procedures below. Our completed safety form can be found here.

Specific Safety Concerns

Laboratory Safety: Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.

Environmental Safety: If any biological materials escape from the lab there is a risk of transfer of antibiotic resistance from our engineered strains into other organisms. Furthermore, dissemination of the Entericidin B peptide could potentially affect the microbiological ecosystem present at the release point, but we plan to secure the release of the EcnB peptide through the use of our engineered fish tag system.

Flavobacterium:Flavobacterium psychrophilum, while a dangerous pathogen for salmonids, carries little to no negative health consequences for humans. It has been classified as a Biosafety Level 1 organism according to the NIH, and is thus essentially harmless to the human population. It is safe to work with and does not require special laboratory protocols beyond those used to work with E. coli.

Entercidin B: Entericidin B is a bacteriolytic and thus is toxic to bacterial cells. The peptide has been found in the human gut , indicating that it is not of harm to the human body at the dosages used in the product. Working with EcnB is thus a relatively risk-free endeavor and does not require special safety procedures.

Safety Protocol

Wet Lab: All lab members wear nitrile gloves, closed-toe shoes, and use eye protection when working with volatile chemicals or UV light. Gloves are replaced and hands are washed immediately after using ethidium bromide or any of the metal solutions. Members work in small groups to ensure if any harm comes to one, others are there to assist. When working with a new reagent or piece of equipment, a faculty lab manager or experienced member is always present to assist.

There are taped off, designated areas for working with ethidium bromide. These areas are cleaned before and after work and are the only areas the solution may touch. All toxic waste is placed in a specialized receptacle and is picked up and disposed of by Cornell Environmental Health and Safety.

All disposables that come in contact with biologics are disposed of in biohazard waste. The lab space also contains sharps containers for disposal of all sharps that contact biological material. All biohazard waste is autoclaved and transported to the building's centralized waste facility where it is disposed of as regulated biological waste.

We maintain 2 copies of MSDS's for every chemical we use in the lab: one for our own records and one for the lab manager and users of the lab space who are not part of our team. The lab is equipped with flame-retardant benches, spill kits, safety showers, eye-washes, and fire extinguishers.

Dry Lab: We use the Emerson Machine Shop for fabrication; each of the dry lab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the dry lab subteam was trained in the safe usage of the milling machine and the metal lathe.

All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed-toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects.

Training and Enforcement

Training : All team members who work in the wet lab must complete Cornell EH&S general lab safety and chemical waste disposal courses prior to the onset of work. These courses set specific guidelines and are the standard requirement for work in a biosafety-level 1 lab at Cornell. Additionally, all team members must complete a lab orientation session with the manager of the BME instructional lab, Dr. Shivaun Archer. During these sessions, Dr. Archer familiarizes new members with the safety equipment and procedures specific to the labspace in which we work.

Prior to the onset of work for the year, all new members are required to go through a safety training program. During this program, safety officers reinforce safety procedures learned during the EH&S courses, discuss safety protocol pertaining to specific chemicals with which we work, and ensure all lab members fully understand all safety procedures.

Safety Officers: The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the Institutional Biosafety Committee to ensure proper equipment usage.

These team members are responsible for discussing the proposed work plan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the Environmental Health & Safety guidelines. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.

Enforcement: Team members who violate safety rules are required to work under the supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of performing the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.