The Importance of ARROWE

Upon the commencement of this project, we spoke to two WNS experts: Dr. Fuller, an ecophysiologist and bat expert with Texas Parks and Wildlife, and Jennifer Smith-Castro, a recovery biologist with U.S. Fish and Wildlife Services. Each provided valuable perspectives on issues related to WNS and emphasized the importance of bats. Their insightful deliverables helped shape our experimental process and change our approach for developing a viable biosensor.

Both Dr. Fuller and Mrs. Smith-Castro have had hands-on experience with detecting WNS and are familiar with the symptoms of the syndrome. These two professionals explained the instrumental role that bats play in our ecosystem, and the importance of detecting WNS. They expressed to us that bats provide an innumerable amount of ecological services such as saving farmers an estimated $3.7 billion on pesticides and crop damage each year [1]; acting as the sole pollinating-agent for the agave plant; and having over 300 species of fruit dependent on them for pollination [2]. These factors, among others, implicate the large-scale harmful impact tied to WNS, and highlight the importance of detection and prevention of dissemination.

In our multiple conversations, Dr. Fuller specified that the detection of WNS is important in all locations where bats dwell, regardless of the species. There are three specific species that are susceptible to White Nose Syndrome: the Myotis velifer, Perimyotis subflavus, and Eptesicus fuscus species. However, bat species that are not directly vulnerable can still become vectors that spread the disease and harm other bat populations, so it is important to test each location regardless of the bat’s susceptibility. Dr. Fuller’s adamant remarks represent the importance of quick and efficient detection of WNS to stop it from spreading.

Iterative Engagement

In our first meeting, Dr. Fuller provided us with some pitfalls to be aware of when creating a biosensor for White Nose Syndrome. A prevailing obstacle was that caves can be hard to reach and require experts to squeeze into small spaces, so carrying a bulky set of instruments or collection tools would not be feasible. In addition to this, freezing samples or incubating bacteria or samples in the field is also not a viable course of action because the equipment involved would be too cumbersome. From this we had to alter our original plan that involved onsite rapid testing. We now plan to have this biosensor compatible with inexpensive and more standardized equipment, such as our biosensor and a -20 ºC refrigerator, so that more labs are able to conduct the testing.

Our iGEM team had some time to talk with an Austin Bat Refuge team member, Lee Mackenzie. Austin Bat Refuge organization helps rehabilitate injured and sick bats by sheltering and fostering them in their facility. Mr. Mackenzie had an apprehensive view on introducing genetically modified bacteria into the environment. But after discussing with them that the testing would be done in a controlled lab, they were open to hearing about what they claimed to be a project of “ingenuity”. Through our discussions with various professionals, and hearing many misgivings of onsite testing, we changed our plans and decided it would be best to have this test be performed in a controlled lab-setting rather than in the field using a minimal amount of environmental sampling.

The Innovative Aspect

In our second meeting with Dr. Fuller, we learned that the current processes for detecting WNS can take many months because it requires specialized equipment and procedures. These procedures are extremely difficult to replicate in uninitiated labs lacking the proper equipment and knowledge. As a result, there are only a few labs in North America that are currently able to detect WNS, causing delayed response times. Because of this, we are designing our biosensor with the hope that it will be used in non-specialized labs and provide quick and standardized results. The timely detection of WNS will help with future treatment and limit the propagation of the disease via human carriage. See our Implementation page to learn more.

Once we learned about the specialized lab, we reached out to Dr. Jeffrey Foster, a well informed researcher on P. destructans detection, the pathogen responsible for the infection of WNS. Dr. Foster works at one of the few specialized detection labs and explained to us the quantitative PCR (qPCR) procedure used in his lab. He discussed the advantages of using qPCR testing to detect if a sample of sediment has P. destructans present was the accuracy and sensitivity of the method. However, when we explained our project, Dr. Foster was intrigued by the innovative method that our project utilizes, and commended our plasmid design. He was even a little apprehensive to endorse our project because, if it is successful, it will be in direct competition with his lab.

When speaking with Dr. Foster, we asked some questions to confirm some of our speculations and buttress the feasibility of our biosensor. We were worried that the method we used to identify our unique target sequence of P. destructans would not be good enough to stimulate our chassis. When asked, Dr. Foster confirmed that our biosensor should be able to detect P. destructans from environmental soil and sediment samples, and validated that the BLAST method we used— to identify a unique target sequence of P. destructans— was fit for our cause. He described that he had never encountered a miscue or false positive with regards to P. destructans eDNA detection in his two decades of working with it. This came as good news, as we were able to progress with our project with more confidence in our approach.

Identifying our Track

Our iGEM team decided to use Acinetobacter baylyi (ADP1) as our primary chassis organism for a variety of reasons. Revered for its natural competency, ADP1 aligned with our goal of detecting sequences of environmental DNA (eDNA), in addition to this we have ample knowledge on how to handle the organism and experiment novel ways to use the bacteria because we are working under one of the leading ADP1 professionals, Dr. Jeffrey Barrick. Moreover, ADP1 is in the early stages of growing its popularity with synthetic biologists, so by using it as our chassis organism we would help progress the widespread usage and engineering of the bacteria.

Upon talking to WNS researchers and bat experts alike, we realized that our project had more potential applications, and could have a greater impact on a larger community, in the foundational advance of synthetic biology. So to grasp a better understanding of where to take our project, we began deliberating with ADP1 professionals outside of our mentor Dr. Barrick. These professionals provided insight and corroboration for our cassette design plans and proffered future steps/improvements for our project. In these meetings, we were also able to share new ways to use ADP1 with some of these scholars.

Examples of ADP1’s growing versatility come in many forms. We had the opportunity to discuss our project design with an iGEM team from Australia that is working with ADP1 genes to degrade aromatic compounds. The 2022 Newcastle iGEM team reached out to the global slack community looking for teams working with ADP1, and we responded. In our fruitful discussion we informed them on a feasible way to use the organism as not only a degradation system but also as a biosensor to detect the presence of aromatic compounds. This simple act of exchanging and sharing new knowledge with other researchers in the field is demonstrative of foundational advance in synthetic biology.

Advice and Knowledge

In the multiple meetings pictured below, we talked with two ADP1 professionals, Dr. Bradley Biggs and Dr. Robert Cooper, about the feasibility of our project and how we are attempting to detect White Nose Syndrome using ADP1’s natural competency characteristic. Both men have had prior experience with ADP1 and they shared some of their most recent research. Dr. Biggs used ADP1 to test the efficiency of different promoters and parts, although he is not working with ADP1 at the moment, he is very optimistic about the organism and looks forward to working with it in the future. Conversely, Dr. Cooper is currently working with ADP1 as a biosensor for cancer detection in the environment and animal GI tract. Dr. Cooper is employing a system that uses the same concepts as ours, leveraging the natural competency of ADP1 with flanking homology arms in tandem with a repressor.

In our meeting with Dr. Biggs, he gave us a stamp of approval on the design of our construct and the method we used to approach this problem. He also informed us on a host of resistance genes that could be more readily knocked out, “fixing” the broken chassis in the presence of P. destructans. This would aid in the positive selection marker indicating the presence of the pathogen. From that conversation we now have a direction for future adjustments that could be done to our biosensor, with little difficulty, to help increase the sensitivity and accuracy of the test.

We discussed with Dr. Biggs about how our work with ADP1 ties into the contribution to the synthetic biology field as a whole. He explained that ADP1 is a very promising organism in many fields of synthetic biology. And due to its special abilities, ADP1 has a very high likelihood of being a main organism used in the synthesis of DNA and the creation/insertion of genetically engineered plasmids, supplanting E. coli. Dr. Biggs discussed that ADP1 also has the potential to be a host for synthesizing chemicals that act as a fuel source for other systems, and act as an effective biosensor for DNA because it is easy to manipulate relative to other host organisms used in the lab. Dr. Biggs lauded our work, claiming that by using ADP1, we are assisting in expanding the popularity of ADP1 as a host organism in synthetic biology, and we are broadening ADP1’s credibility as a host organism by demonstrating its versatile nature.

Upon showing Dr. Cooper diagrams of our design for our plasmids, he supported the design, and proclaimed it looked “like a sound system”. Our cassette was similarly designed to what he has been doing in the lab. Dr. Cooper offered further comments on possible future steps to improve the expression of Yellow Fluorescent Protein (YFP) in our system, such as finding stronger ribosome binding sites and stronger promoters— these were insights that could influence the future steps of the project.

With direct respect to foundational advancements in synthetic biology, Dr. Cooper was curious about the repressor that we were using because the repressor that he had in his system turned out to be toxic to the growth of ADP1. We informed Dr. Cooper of the CymR repressor part that we were using, and he was very enthusiastic about it. This simple act of informing other scientists about the specific parts we used in ADP1 is an explicit demonstration of foundational advance.

Everything considered, our meeting with Dr. Biggs and Dr. Cooper fortified our understanding of ADP1 as a dynamic organism in the field of synthetic biology, and reinforced our decision in choosing ADP1 as our host organism. We were able to tell another scientist about a non-lethal repressor that we found to work fine in ADP1, and from this we are helping to advance ADP1 as a chassis organism. Dr. Biggs bolstered our iGEM team’s experiment and was excited to see young scientists working with such a promising organism.

Keep Austin Batty

Our iGEM team visited the Austin Bat Bridge, in downtown Austin, and interviewed some observing civilians. From these interviews we discovered the importance of the Bat Bridge to Austin. Aside from the environmental role that bats play in our environment, they are also a determining factor of what makes Austin unique. One civilian explained that having “nature in the heart of the city” is what makes Austin special, and to them bats had a special part in the soul of the city. Substantiating this claim was the fact that the majority of the people we interviewed came from out of state and visited the bat bridge because it was described as a top-spot to visit in Austin by various pamphlets, visitor maps, and websites. Each person we interviewed claimed our project to be a righteous pursuit. One civilian, who wished to remain anonymous, supported our research enthusiastically saying, “of course, we want to save the bat population!”

As UT Austin’s 2022 iGEM team, we strongly believe that what we are doing is important on two fronts. We are contributing to growing the realm of science by using a relatively nascent organism in a creative way, while also helping a species that is important to both the ecosystem and the hearts of many.

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