Team:CCA San Diego/Safety

Overview

Team CCA_San_Diego is in full compliance with iGEM’s safety and security guidelines. Throughout the process of designing, constructing, and implementing our project, we have continuously prioritised safety above all else, whether that be in the form of special procedures, practices, or materials.

All experiments were conducted at the J. Craig Venter Institute (JCVI). They were thoroughly researched during the planning process, and, with the guidance of the scientists at the lab, were approved - and modified, if necessary - prior to execution.

Additionally, team members working in the lab underwent an extensive safety training, in which they learned standard procedures and practices to ensure an optimal environment that would be conducive to secure and effective scientific experimentation.

Our project also aims to provide a safer work environment for researchers in the field of collagen research and patients who will receive transplants with new organ technology by replacing toxic industrial chemicals with nontoxic alternatives. Our team conducted research on using genipin as a crosslinking reagent instead of the standard chemical glutaraldehyde.

Choosing a Chasis

Prior to our experimentation in the lab, we were faced with several decisions regarding the microorganisms we would use in our experiment. For an easy and effective Gibson assembly, a standard E. coli strain could be used - however, due to our goal of creating a novel template capable of gap-repair cloning of multiple fragments, we wanted to find a competent and safe yeast strain.

In addition, as the protein we wished to express was coding for a surface protein involved in cell adhesion from Streptococcus pyogenes, a BSL-2 pathogen, we decided to order the repetitive collagen sequence instead of extracting directly from the host organism. Although this proved very difficult due to the many repeats of the collagen protein, we prioritized our laboratory safety and aimed to develop a protocol that would be safer for continued research.

Lab Safety

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Prior to any actual lab work, a certification - obtained through extensive safety training - for all team members involved was mandatory, and the following topics were covered:

  • Hygiene: washing hands, disinfecting surfaces, no food or drink allowed, etc.
  • PPE: lab coats, gloves, eye protection, masks, close-toed shoes, etc.
  • Biohazard disposal: containers, special areas for disposal, etc.
  • Storage: proper labelling, understanding the purpose of different freezers, etc.
  • Handling: how to handle hazardous chemical materials, etc.
  • Emergency: what to do in the case of spill or contamination, etc.

In addition, all procedures were thoroughly researched and approved before their implementation.

Project Hazards

Glutaraldehyde

Glutaraldehyde was used as a crosslinking reagent for the different products created with the yeast-expressed collagen. If handled improperly, the chemical can be extremely toxic, potentially causing throat and lung irritation, asthma, nose irritation, eye irritation, rashes, staining of the hands, hives, headaches, nausea, and more (CDC 01). When it comes to maintaining a safe environment, the requirements (as explicitly defined by the Center for Disease Control, or CDC) include the following:

  • Using local exhaust ventilation
  • Keeping glutaraldehyde baths under a fume hood when possible.
  • Using only the amount of glutaraldehyde needed.
  • Avoiding skin contact.
  • Washing gloved hands after handling.
  • Wearing goggles and face shields.
  • Sealing or covering containers containing glutaraldehyde solutions.
  • Attending safety classes to understand how to properly address the risk of exposure.

To ensure safety in our lab, we followed all guidelines; all experimentation with glutaraldehyde (classified as biosafety level 2) was performed under a fume hood, and thorough precautions were taken when labelling containers and storing them in the appropriate freezers.

Porcine Skin

Before the yeast-expressed collagen was developed, in order to experiment with the procedures necessary to create our final products (thread, gel, and felt), we used collagen extracted from porcine skin as proof of concept.

In order to handle the porcine samples, all surfaces and tools (razors and scissors) were disinfected prior to procedures with ethanol, the laboratory standard disinfectant commonly used in surgical instances due to its ability to eliminate corrosive residues (Stanford). Surfaces were cleaned with 10% bleach after the experiment and filtered collagen was stored in a separate container in a 4oC fridge.

Acids

When using strong acids like acetic and hydrochloric acid, all experiments were performed under the fume hood.

Replacing Glutaraldehyde

Glutaraldehyde is the standard reagent for cross-linking and is commonly found in many laboratories and working spaces. Although scientists are advised to take safety precautions, such as using a fume hood and wearing adequate personal protective equipment, it still remains a safety hazard in the lab and risks cytotoxicity when used in biomedical products. Because of this, our team conducted extensive research on genepin, which is a natural crosslinking reagent derived from Gardenia jasminoides ELLIS(Yoo et. al, 2011). Genepin is non-toxic and has had various roles as herbal medicines in Eastern cultures. Additionally, studies have shown that genepin is not only biocompatible, but is also effective in crosslinking as glutaraldehyde, making it a much better alternative on standards of cyto- and genotoxicity.

Reducing Transmission of Animal Diseases

As the need for collagen for various purposes is rising, the risk of variant Creutzfield-Jakob's disease (vCJD), more commonly known as Mad Cow’s Disease, also rises. Most industrial collagen is extracted from the bovine of animals, namely cows and pigs. vCJD is transmitted through digestion or contact with infected cow bovine. Thus, by creating bacterial collagen, the risk of transmission decreases greatly because the yeast does not come into contact with cow bovine. This is especially important because collagen has risen in its biomedical applications recently and due to the nature of the disease infected collagen can be dire to the patient.

Final Result

Overall, our final products - thread, gel, and felt - are intended to be extremely safe for practical use, as they are intended for medical purposes (see more at our Proposed Implementation). Our project does fulfil that goal; however, there still exists the risk that accompanies the use of glutaraldehyde in our protocols, which we hope to substitute for both our experiments and those of future researchers.

References

CDC. (2001). Glutaraldehyde: Occupational Hazards in Hospitals. Department of Health and Human Services, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health.

Stanford. (n.d.). Comparing different disinfectants. Stanford Environmental Health Safety iCal. https://ehs.stanford.edu/reference/comparing-different-disinfectants.

Yoo, Jae Suk et al. “Study on genipin: a new alternative natural crosslinking agent for fixing heterograft tissue.” The Korean journal of thoracic and cardiovascular surgery vol. 44,3 (2011): 197-207. doi:10.5090/kjtcs.2011.44.3.197