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The Autonomous Microbial Genosensor (AMG)

Researcher Contact: Matthew Smith
The AMG represents a fully automated system for in situ genetic analysis. Designed for field deployment, the AMG combines systems for sample collection, filtration, cell lysis, RNA extraction/purification/concentration, gene amplification and data transmission. The AMG has been designed and built at the Center for Ocean Technology by adapting and merging molecular biology protocols with a combination of mechanical and electrical engineering, off-the-shelf parts and in-house fabrication. Development and testing of the AMG has been performed on site in the Bio-Molecular Sensor Laboratory (BSL) (visit the BSL). While the prototype AMG is configured to detect the Red Tide causing dinoflagellate Karenia brevis, the goal is to develop an auto-analyzer that can be used to monitor for virtually any organism for which sequence information is known by varying the cell lysis procedures and gene primer/probe combinations. The AMG has application toward environmental monitoring of harmful algal blooms, water-borne pathogens, biowarfare agents or organisms of ecological interest.
Sensor Description
The AMG is housed in a 22cm x 122cm (diameter x length) anodised aluminium pressure vessel. A separate 19cm x 35cm (diameter x length) pressure vessel houses four lithium ion batteries that power the unit. The current prototype sensor is designed for subsurface deployment for 3 days where it samples between 30-50 ml of water and performs 12 assays (including negative controls).

Individual samples are processed sequentially through a series of two rotating wheels. The first wheel contains custom filter/RNA purification columns and is responsible for sample concentration, cell lysis and RNA purification/concentration. The second wheel performs RNA amplification. The operation of the AMG can be divided into 3 distinct modules – Sample Collection/Preparation, Nucleic Acid Amplification and Data Transmission.
Image showing the sample collection/purification and reaction wheels. The upper wheel contains custom filter/RNA purification columns and is responsible for sample concentration, cell lysis and RNA purification, the second wheel is dedicated to gene amplification. A series of fluidic injectors interact with the two wheels to introduce the sample or buffers for RNA purification or gene amplification. • Image showing the sample collection/purification and reaction wheels. The upper wheel contains custom filter/RNA purification columns and is responsible for sample concentration, cell lysis and RNA purification. The second wheel is dedicated to gene amplification. A series of fluidic injectors interact with the two wheels to introduce the sample or buffers for RNA purification or gene amplification.
Sample Collection/Preparation
The (AMG) uses a syringe pump to sample and filter water through custom sample collection/RNA purification columns. Following filtration the seawater is returned to the environment. The sample collection/purification columns have been designed and built in-house and are based around standard micropipette tips and a solid phase silica matrix. Cells captured on the filter then undergo chemical lysis and RNA is purified using a Boom (1) style extraction using buffers obtained from Stratagene (La Jolla, CA, USA). Following purification, RNA is eluted using a custom elution/NASBA reagent buffer. An aliquot of the eluted RNA is then taken for analysis. All reagents used in sample preparation are contained in a waste bag in the AMG, and are safely disposed of in the laboratory post-deployment.
Image showing the AMG fluidic management system. Reagents required for sample preparation and nucleic acid amplification are housed in individual reagent bags. Fluidic manipulations are dispensed through custom fluidic manifolds using Instec mini peristaltic pumps and Burkert micro-valves. Volumes of solutions moved by the system have been calibrated as a function of pump speed, input pulse width frequency and valve timing • Image showing the AMG fluidic management system. Reagents required for sample preparation and nucleic acid amplification are housed in individual reagent bags. Fluidic manipulations are dispensed through custom fluidic manifolds using Instec mini peristaltic pumps and Burkert micro-valves. Volumes of solutions moved by the system have been calibrated as a function of pump speed, input pulse width frequency and valve timing.
RNA Amplification
The AMG detects the presence of a target organism at a genetic level by using Real-Time Nucleic Acid Sequence Based Amplification (RT-NASBA). NASBA is performed using a custom single reagent sphere that has been developed by a collaborative effort between the Systems Technology Group at COT and Life Sciences Inc (St Petersburg, FL, USA). This one-tube reaction uses lyophilized NASBA spheres containing primers, molecular beacons and the trimeric NASBA enzyme cocktail. The reaction is initiated by re-suspending the spheres with a custom reaction buffer prior to addition of the RNA and heating to 41˚C.
Image showing the reaction/detection wheel. The wheel contains blank PCR tubes for elution of RNA, and tubes containing the single NASBA reaction spheres. Real-time. Detection of NASBA amplification is performed in a heated reaction/detection chamber. The chamber consists of an infra-red heater and detector, detection is achieved using a low-cost, LED and photodiode. • Image showing the reaction/detection wheel. The wheel contains blank PCR tubes for elution of RNA, and tubes containing the single NASBA reaction spheres. Real-time. Detection of NASBA amplification is performed in a heated reaction/detection chamber. The chamber consists of an infra-red heater and detector, detection is achieved using a low-cost, LED and photodiode.

The primer/probe set used for the detection of K. brevis was developed in the laboratory of John Paul. Integration of the K. brevis assay into the AMG has been performed at COT. Laboratory based K brevis extraction and amplification (2) and the automated AMG extraction and amplification has been shown to produce comparable results.
Data Transmission
Data is transmitted to shore based operations using a 2.4 GHz ISM band
 wireless connection that Complies with 802.11b (Wi-Fi). This system has a range of 1000+ feet outdoors and is scalable in range with the impending WiMax
standard and Interhop-based Mesh Networks. The wireless communication module can be mounted as part of the AMG electronics or as an external standalone unit. When mounted externally, the wireless module is integrated into the floatation buoy. The externally mounted wireless system provides the additional advantages of having a self contained power supply that allows constant data transfer as well as providing a second channel for data transmission from a second sensor such as a CTD. Using 802.11x wireless connections, we have demonstrated a low cost high bandwidth heterogeneous network for embedded sensor communication. The AMG has been tested as part of this embedded sensor array, which effectively linked the AMG with diverse sensors for physical and chemical monitoring. These adaptive sensor arrays will prove integral in developing nowcast/forecast ocean models.
AMG technician James Wilson with an AMG buoy/wireless transmission module during a test deployment/embedded sensor network in Tampa Bay, FL 2005. • AMG technician James Wilson with an AMG buoy/wireless transmission module during a test deployment within an embedded sensor network in Tampa Bay, FL 2005.
AMG Field Deployment
The next phase in the development and testing of the AMG hardware is in progress, we are currently investigating, characterizing and fine-tuning the systems and operation of the AMG by deploying the unit in Bayboro Harbor adjacent to COT. Following this system optimization the unit will be deployed in the greater Tampa Bay area to monitor for Red Tide (K. brevis) blooms.
COT staff during a field deployment trial of the AMG system in Bayboro Harbour, St Petersburg, FL, 2005. • Field deployment of the AMG. COT staff undergoing a field deployment trial of the AMG system in Bayboro Harbour, St Petersburg, FL, 2005.
References

(1) Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa (1990). Rapid and Simple Method for Purification of Nucleic Acids. Journal of Clinical Microbiology 28:495-503.

(2) Casper, E. T., J. H. Paul, M. C. Smith, and M. Gray (2004). Detection and quantification of the red tide dinoflagellate Karenia brevis by real-time nucleic acid sequence-based amplification. Applied and Environmental Microbiology 70:4727-4732.
Presentations
Matthew C. Smith, Andrew S. Farmer and David P. Fries (2005). In Situ Sensor Technology. International Association of Food Protection's 2005 Annual Meeting, Baltimore, 14-17 August, 2005. Oral Presentation.

Matthew C. Smith. Andrew S. Farmer, John H. Paul, Sunny Kedia, Scott A. Sampson, David Ritchey, George Steimle and David P. Fries (2005). Sensors in the Sea, Sky and Beyond. 105th ASM General Meeting, Atlanta, Georgia, 5-9 June, 2005. Poster Presentation

Smith, M. C., Farmer, A. S., Neumann, O., Casper, E. T., Gonzales, G., Patterson, S., Paul, J. H., and Fries, D. P. (2005). The Autonomous Microbial Genosensor (AMG) - An Open Platform Autonomous Genetic Sensor For Real-Time Detection Of Marine Microorganisms. American Society of Limnology and Oceanography, 2005 Aquatic Sciences Meeting, Salt Lake City, Utah, USA 20-25 February, 2005. Poster Presentation.

D. Fries, H. Broadbent, G. Steimle, M. Janowiak, X. Fu, A. Cardenas, A. Farmer, M. Smith, T. Batheja, S. Samson, E. Kaltenbacher, J. Paul, S. Ivanov Marine MEMS: Applications and Opportunities, Micro and Nano Technology at PIM 2004, Manaus, Brazil, September, 2004. Oral Presentation.

Smith M. C., Farmer, A. S., Fries, D. P., Casper E. T., Gonzales, G. & Paul, J. P. (2004) Monitoring the seas: the Autonomous microbial genosensor. 104th ASM General Meeting. New Orleans, Louisiana, 23-27 May 2004. Poster Presentation.

Smith, M. C., Paul, J. H., Fries, D. P., Casper, E. T., Farmer, A. S. & Gonzalez, G. (2004). Towards Autonomous in situ Microbial Monitoring – the Autonomous Microbial Genosensor(AMG). American Society of Limnology and Oceanography and The Oceanography Society 2004 Ocean Research Conference. Honolulu, Hawaii, 15-20 February. Oral presentation.

Farmer, A. S., Fries, D., Paul, J., Smith, M. C. and Gonzalez, G. A. (2004). A Tunable Automated Nucleic Acid Biosensor. American Society of Limnology and Oceanography and The Oceanography Society 2004 Ocean Research Conference. Honolulu, Hawaii, 15-20 February. Oral presentation.

Matthew C. Smith, John H. Paul, David P. Fries, Erica T. Casper, Gino Gonzalez & Andrew S. Farmer (2003). From Star Trek to the Sea: Development of an Autonomous Microbial Genosensor. American Society for Microbiology, Southeastern Branch Meeting, Athens, Georgia. 30 October – 1 November. Oral presentation

John H. Paul, David P. Fries, Matthew C. Smith, Erica T. Casper & Andrew S. Farmer (2003). Towards automated in situ microbial monitoring - the autonomous microbial genosensor (AMG). Biocomplexity in the environment awardees meeting, Arlington, Virginia, 15-17 September. Poster presentation

J. Paul. (2003). Marine Microbial Biocomplexity: Viromics and an Autonomous Microbial Genosensor. Biocomplexity in the environment awardees meeting, Arlington, Virginia, 15-17 September. Oral Presentation

J.H. Paul (2003). Application of NASBA to the design of an Autonomous Microbial genosensor. The Next generation of in situ biological and chemical sensors in the ocean. 13-15 July, Woods Hole, MA Oral Presentation.

Matthew C. Smith, John H. Paul, David P. Fries & Andrew S. Farmer (2003). Application of a Sensitive Real-Time NASBA Assay for the Detection of Synechococcus spp. in an Autonomous Microbial Genosensor (AMG). American Society for Microbiology 103rd General Meeting. Washington DC, 18-22 May. Poster presentation

Matthew C. Smith, John H. Paul, David P. Fries & Erica T. Casper (2003). Developing NASBA for Real Time Detection of Microbial RNA Targets in the Marine Environment. American Society of Limnology and Oceanography Aquatic Sciences Meeting, Salt Lake City, Utah, 8-14 February. Oral Presentation

Matthew C. Smith, John H. Paul, David P. Fries & Andrew S. Farmer (2002). Development of an Autonomous Microbial Genosensor (AMG). Florida Marine Biotechnology Summit III, Fort Pierce Florida. 7-8 October. Poster presentation.

J.H. Paul. (2002). Single target detection technologies. Ecogenomic Workshop, Kansas City, MO. 9-11 May 2002.

J. H.Paul and D. Fries. (2002). An Autonomous Microbial Genosensor. ONR Joint Review of Technology Applicable to Mine Countermeasures and Associated Missions. Panama City, FL, 6-7 April.
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