InterLab Human Practices Collaborations

Research

Professional Input

Tiffany Black from the Florida Fish and Wildlife Conservation Commission provided some insightful feedback on our project. In her email, she highlighted how education is the key to protecting wildlife. This enables people to appreciate the wildlife around them and to be more likely to protect their environment. However, she described that this is not as easy as it sounds, since people “want to have their cake and eat it too”. A viable alternative to LAL testing may be beneficial to horseshoe crab populations but in consequence may be very expensive. The same applies for the current LAL testing practices. The horseshoe crab blood harvest is currently the most efficient way to achieve endotoxin detection through LAL.

Finding a balance between these two aspects of LAL testing, cost and environmental consequence, is extremely important. If the decline in the horseshoe crab population is so great that their blood can no longer sustain the biomedical industry, then the industry will have to find a more expensive alternative with the consequence of decimating the horseshoe crab population as well.

Black also mentioned the controversies of LAL testing. She stated that biomedical companies do their best to minimize the mortality rates of horseshoe crabs during harvest but the average mortality rate in North America is still about 15%. She also touches on the animal testing to determine the effectiveness of drugs on humans. There have been ethical issues about this practice, but as long as the only alternative is human testing, it is a necessary practice. Black then discusses the illegal capture of horseshoe crabs. She voices the struggles of getting a species protected; in order to achieve protected status, data must be gathered and stakeholders must show interest.

Black ends by bringing it all back to the importance of education. She relays the story of a woman from the Delaware Bay area who loves horseshoe crabs. This woman saw a presentation on horseshoe crabs and was told that they could be saved by flipping them over if they were stranded. She went on to save 25,000 as of 2009 and saving more to date. If the environmental education of one woman made such a big difference, then the impact of widespread education could be enormous.

“So, in closing to you, my friends in this ‘environmentally conscious organization’ …in so many cases, things have to start in your homes, in your heads and in your hearts. You have the power to try to become engaged in your own impacts on the planet. We used to say ‘recycle, reuse, reduce.’ My personal favorite is ‘reduce, reduce reduce.’ Reduce your plastic. Reduce your fossil fuel use. Reduce your water use. Read labels, and LEARN about your wild places and how to fit into them with a lessened impact. In just trying, you are doing so much. In wanting to even entertain the alternative LAL idea, you could go on to do great things.”

We also got responses and feedback for our project from various other experts:

Dr. Thomas Novitsky, the CEO of Cape Cod Biosystems, Inc. who worked with Dr. Stanley Watson who learned how to make LAL from its discoverer, Dr. Jack Levin.
Mark Botton and Paul Shin, the co-chairs of the IUCN SSC Horseshoe Crab Specialist Group.
Dr. David Hussong and Melissa Stappen of ValSource, LLC who wrote an article called “The LAL Assay for Pyrogen Testing of Parenteral Products: Evolution & Challenges”.
Dr. NG Wai-Chuen from the Hong Kong Agriculture, Fisheries and Conservation Department that enforces regulations on plant, pesticide, and animal controls, oversees the efficient production of fishery and agricultural produce, and safeguards ecological integrity.
Brenda R. a drug informational specialist for the FDA Division of Drug Information, which is composed to healthcare professionals and pharmacists who provide education to the general public as well as targeted audiences such as small businesses or industries.
Dr. Daniel Ziegler, Director of Bacillus Genetic Stock Center at the Ohio State University, gave us guidance on whether to use b. Subtilis or b. Cereus, which plasmid to use, where to insert the fragments.

Education

Seattle’s Mini Maker Faire Booth

On August 19, Seattle held its annual Mini Maker Faire which showcases the work of tech creatives, scientists, and other intellectuals in the Seattle area. At the Mini Maker Faire, the community lab that our team works out of, SoundBio Lab, were one of the featured makers at the event. As the SoundBio Lab was planning on hosting an educational, outreach activity at the Faire, we also thought it would be fitting to help talk with the members of our Seattle community and discuss the topics of synthetic biology as well as more specific concepts that our project is working on addressing. We had many conversations with Seattle citizens about the importance of health care and specifically having access to sterile, safe medical equipment, protecting different animal species, and the significance of biotechnology in our daily lives.

Most memorably, the people we talked to were intrigued to learn that LAL testing is used in Seattle’s own hospitals and companies. Rim Bio, a Seattle-based company that produces single-use bioprocess containers performs LAL testing on their products to ensure that customers receive clinically sterile products[1]. Namely, Rim Bio uses the Charles River Endosafe® Test that harvests horseshoe crab blood. However, Charles River unique compared with other LAL companies as it is focused horseshoe crab conservation as stated on their website:

“Once the crabs are brought into our lab, they are carefully inspected by trained employees who determine the animal’s health and maturity. After careful collection of a measured amount of blood, the crabs are returned unharmed to their natural habitat within the same day, and their blood volume rebounds quickly. These practices have allowed Charles River to achieve an industry-leading survival rate of donor animals”[2]

We also were able to connect with a lot of parents who were visiting the booth with their kids as we talked to them about Seattle Children’s Hospital, one of the best children’s hospitals in the country. Many families in the Washington area have received life-saving treatments at Seattle Children’s and like Rim Bio, Seattle Children’s also uses LAL testing for many of their products and procedures [3]. These families were surprised to hear that some of these life saving procedures were done at the expense of horseshoe crab bleeding, and were excited to learn about our project to synthetically produce Factor C.

Gasworks Park “Learn About DNA Day”

As part of the Pacific Northwest iGEM Meetup, we held an outreach event at Gasworks Park, a popular spot for families to visit. Our goal was to educate the public about the significance of our research problem, as well as spark interest towards the growing field of synthetic biology as a whole.

Our outreach activity was primarily targeted towards young children. We created a “Code Your Name with DNA” activity, where children were taught about the structure of DNA, what DNA is used for, and some of the amazing things DNA can do in synthetic biology. This activity began with students writing out their name and matching the letters of their name to a special key. In this key, each letter of the alphabet is ‘coded’ by a set of 3 nitrogen bases, similar to the formation of the polypeptide chain in RNA translation, and the kids created a bracelet that corresponds to their name and is reminiscent of the double stranded structure of DNA.

While half of the human practices team was working directly with youth, the other half of the members spoke with parents and adults that were passing by. By connecting with adults, who have a large influence in our community, we were able to describe to them the dire situation of horseshoe crabs, and potential future shortcomings in the healthcare field due to the current LAL testing. Then, we explained our proposed solution with the synthesis of Factor C in a straightforward way that nearly anyone could understand. Through teaching others about this issue, we were also able to learn from the public as well. For example, several of the parents asked us if the expenses involved in switching to synthetic endotoxin detection will reduce its accessibility to consumers, especially in low-resource settings. Prior to this, we knew our solution had to be less expensive than extracting the blood of horseshoe crabs, but by speaking directly with those whose lives will be altered by our solution, we how critical the monetary aspect of our project was. Thus, the direction of our project was slightly shifted towards not just finding an alternate solution to current LAL tests, but also on making it accessible to all people by reducing costs.

Design

Dr. Daniel Ziegler, Director of Bacillus Genetic Stock Center at the Ohio State University, gave us guidance on whether to use b. Subtilis or b. Cereus, which plasmid to use, where to insert the fragments. He advised us to use Bacillus subtilis rather than B. cereus for safety reasons and widely available genetic tools. He also recommended using the strains 1S145 or 3NA as plasmid backbones because they are not large. Large plasmids are usually unstable in B. subtilis because their replication methods often result in long single-stranded intermediates, which can lead to deletions. Dr. Zeigler reminded us to use a gram-positive ribosome binding site. For inducible expression, he provides an example of a xylose-inducible Pxyl promoter, pAX01, that integrates into lacA gene of B. subtilis with erythromycin resistance. For the pAX01 vector, he recommends cloning into 2 of the 4 sites - NotI, SacII, SacI, or BamHI - to do directional cloning. Ultimately, by obtaining feedback from Dr. Zeigler, we were able to positively alter the design of our project.