Overview

To implement our project, our team focuses on two types of end users: White Nose Syndrome (WNS) screening groups and the iGEM Synthetic Biology research community. We hope to provide WNS screening groups with a rapid and robust test for detecting P. destructans in a lab environment, and we aim to provide the iGEM Synthetic Biology community with the tools to use ADP1 as a model organism.

The first type of end users are WNS screening groups, such as Texas Parks and Wildlife, Austin Wildlife, Animal and Plant Health Inspection Services, and World Organization for Animal Health (WOAH). One of the major problems these groups face is that the only available WNS tests are qPCR tests, and the labs qualified to conduct qPCR tests on field samples are only located in Wisconsin and Arizona [1]. This means that test results often come months after samples are sent, which creates a significant delay in data collection on the spread of WNS. Therefore, a simple binary sensor that can quickly and accurately detect the presence of White Nose Syndrome would greatly benefit these screening groups.

The second type of end users are members of the Synthetic Biology community. One of ARROWE's goals is to engineer ADP1 as a chassis organism and to provide the iGEM Synthetic Biology community the tools to create an ADP1-based biosensor. Traditional model organisms, such as E. coli, lack the ability to efficiently take up environmental DNA (eDNA). ADP1's natural competence allows researchers to more easily incorporate new DNA constructs into the ADP1 genome, making it a more desirable model organism for synthetic biology applications [2]. By using a modified ADP1-ISX strain [3] to engineer our three detectors, we aim to demonstrate the versatility and power of ADP1 as a synthetic biology chassis. Additionally, the modular nature of our biosensor circuit makes it easy to reprogram the circuit to detect any DNA sequence of interest, increasing the utility of the ARROWE system. Figure 1 summarizes the possible applications of the ARROWE system.

Proposed Implementation

WNS ADP-1 Biosensor

The White Nose Syndrome fungal organism, P. destrustans, is classified as a BSL-2 organism [4], so only BSL-2 trained personnel and labs may handle samples containing P. destructans. We envision ARROWE testing kits to include a frozen stock of the ADP1 biosensor culture, azidothymidine (AZT, our screening antibiotic), and an instructions manual our team will put together. The instruction manual will contain information on biosensor handling, processing, and data collection once we have fully streamlined our biosensor. 96-well plates and a fluorescence plate reader will be required to interpret the results of the test, which will not included in the kit. In the future, kits could be distributed to labs via a Material Transfer Agreement (MTA) with the University of Texas at Austin. Our team also plans to share the DNA sequence and the strain itself via databases or sites such as GenBank, ATCC, and NCBI upon confirmation of a working biosensor.

To effectively utilize the test, screening agencies should collect several samples (skin swaps from the nose, wings, or soil) from bat populations annually before their hibernation period. Samples should be stored at -20°C and sent to labs equipped with the tests. Tests can be run overnight, and results can be sent to the screening agency the next day. This data can potentially be used by screening agencies to track the spread of P. destructans and the locations of vulnerable bat populations. Figure 2 shows how our team envisions our biosensor being used.

During the development of our biosensor test, we strove to make our test as simple as possible, minimizing the need for expensive lab equipment and trained personnel. While we have succeeded in simplifying this test to a large degree, one challenge is that a plate reader is recommended to confirm the results. Additionally, due to health and safety concerns regarding P. destructans, BSL-2 trained personnel are required to handle samples and process the test.

ADP1 Chassis Strain

Like with our ADP1 biosensor, other labs could acquire our ADP1 strain using an MTA in the future. Our team also plans to upload our sequence to ATCC, GenBank, and NCBI databases. All of our team's engineering documentation and parts can be found on our Engineering and Parts pages.

Safety

References