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ECOHAB: Engineering Upgrades and Field Trials of the Autonomous Microbial Genosensor(2008)

Project Description:
Engineering Upgrades and Field Trials of the Autonomous Microbial Genosensor: This project is part of the Ecology and Oceanography of Harmful Algal Blooms program. Harmful algal can be major catastrophes in terms of economic losses, aquatic organism mortalities, and deleterious impacts on human health. To predict onset of harmful algal blooms, monitor their severity, and to accurately determine their termination, rapid, reliable, and accurate methods are needed to detect HAB species. A major goal is to incorporate rapid and accurate detection methods into ocean observing systems. We have used the ribulose-1,5-biphosphate carboxylase/oxygenase large subunit gene (rbcL) as a molecular tag to detect K. brevis in a prior ECOHAB-funded project. We developed an assay that uses the novel Nucleic Acid Sequence-Based Amplification (NASBA) and molecular beacon technology. NASBA amplification, which is isothermal, is more amenable to field assays and autonomous platforms than PCR, which requires thermal cycling. With prior funding from ONR and NSF, we have incorporated our NASBA-based detection technology into the Autonomous Microbial Genosensor (AMG), the first sensor buoy to perform nucleic acid amplification to detect harmful algae. Based upon our experience with this system we would now like to improve the AMG with several engineering upgrades and embark on a series of field deployments to fully test this system. Our objectives are to: 1. To upgrade the current AMG to a dual channel detection system and other improvements 2. To reduce overall system size and weight by optimizing packaging of the fluidic management system and pressure vessel 3. To build a second AMG unit 4. To determine performance of both units through a series of field deployments. For Objective 1, we will install a second fluorescence channel in the AMG to enable detection of an internal control for quantitation and determination of performance. Alternatively, the second channel can enable detection of a second target species or a different gene (ie. a K. brevis PKS gene). Objective 2 aims to decrease the overall size and weight of the AMG to facilitate easy deployment. Construction of a second AMG (Objective 3) will enable simultaneous deployment and data collection from two sites, which is the main goal of Objective 4. We will manually sample during operation modes of the AMG during field deployments to ensure proper performance, and simultaneous samples will be microscopically counted for K. brevis. The outcome of this research will be an autonomous RNA amplification platform capable of detecting and providing quantitative information on K. brevis populations in near real time. The system will be targeted toward Karenia brevis but with simple modification should be able to target any HAB species. This proposal coincides with the NOAA agency interests described in the RFP: “Development of new methods for measuring HAB cells and toxins, especially those that can be used in ob

Expected Outcome:
Year 1- The Fries engineering group will undertake a program of packaging redesign review and implement packaging modifications for AMG instrument size reduction. This includes creation of a layered fluidic manifold, selection and installation of new materials, and a packaging audit. After these redesigning tasks are complete, the Fries group will commence construction of the second AMG. The Paul lab will initiate the cloning of the rbcL gene ftom K. mikimotoi which is currently cultivated at the FWRI labs in St. Petersburg (next door to the USF CMS). This is accomplished by using PCR primers that we have previously published to amplify the gene ftom DNA ftom K mikimotoi. Once this is cloned it will be sent off for sequencing. Once the sequence is obtained, it will be aligned (using OMl:GA)to other K. mikimotoi, K. brevis, and related haptophyte rbcL sequences. Then, we will select a portion of the rbcL sequence for NASBA primer and molecular beacon design, to develop a specific detection assay for this organism. We will develop our PKS gene expression assay in the following manner. We will develop an IC-RNA sequence and a positive control RNA. The latter will be cloned using RT-PCR ftom a growing K. brevis culture to be provided by FWRI. The clone will be used to generate transcripts to serve as a positive control. The IC-RNA will be designed and made by PCR amplification using two overlapping oligonucleotides. A T7 RNA polymerase promoter will be incorporated into the sequence such that it will enable in vitro transcription without cloning. Using the PKS assay, we will examine the level ofPKS transcription (by NASBA) of Nreplete and N-limited cultures to be provided by FWRI labs. Simultaneously, they will measure the brevetoxin levels in these two types of cultures by ELISA analysis. If time permits, we will repeat this study with phosphate-limited and replete cultures. Year 2- The Fries engineering group will complete the building of the second AMG field unit and validate the analytical performance of the unit on bench. The Paul lab will be working closely with the engineering group to integrate the assays developed in Year I as well as the K. brevis rbcL IC-RNA into the re-designed AMG. This involves developing single shot or single use, two component spheres that contain primers and beacon, as well as coordinated bench level testing of the AMG. Additionally, completion of any unfinished work on the assays or development of the second AMG will occur this year. Year 3 The third year's work will focus on field deployments during bloom and nonbloom conditions. These will occur in the coastal regions of Southwest Florida, contingent upon bloom occurrence. COT personnel will be responsible for predeployment preparation of the AMG, including preparation of reagents for use in the AMG, and securing reagent/enzyme spheres for use. Simultaneous with deployment is sampling for traditional lab NASBA amplification, which will involve Paul lab personnel. Samples will be filtered on site and transported back to the lab for analysis. Results obtained by the AMGwill be compared to that obtained with the lab assay format. Additionally, samples will be taken for cell counts, fixed in Lugol's solution, and counted by FWRI personnel.