Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Imaging, Molecular, and Cellular Analysis
99
High Throughput Fermentation and Cell Culture Device
David Klein (dklein@gener8.net), David Laidlaw, Gregory Andronaco, and Stephen Boyer
Gener8, Inc., Mountain View, CA
The Genomics:GTL (GTL) program requires that multiple microorganisms be grown with high throughput under a variety of carefully controlled-state conditions. Additionally, recombinant clones will also require culture under controlled conditions at high levels of expression, high throughput and fast production turn-around. These endeavors require technology to a) grow specific biomass under well-characterized states b) rapidly identify optimal culture conditions for expression of tagged proteins and complexes, c) rapid scale-up to obtain necessary protein samples, d) express intact protein complexes, and e) grow microbial cells in nonstandard conditions. Towards the creation of high thoughput, controlled-environment instrumentation to meet these challenges, we will present the design and function of a microreactor system with parametric controls comparable to stirred vessel bioreactors. We will further demonstrate that this type of system can be used to enhance the throughput in complex, but routine, workflows.
The system designed is a bench-top, computer-controlled microreactor system. The microreactor uses a disposable cassette (SBS standard) system with 24 individually controlled 10 ml reactors. Each reactor has independent control of temperature, pH, dissolved oxygen. The current configuration covers the following range of operation:
- Temperature (control range of 20 C to 45 C, 0.1 C resolution)
- pH (acidifying and alkalizing from pH 5 to pH 9; 0.01 resolution)
- Dissolved oxygen (0 to over 100 % air saturation, 0.1 to 10% resolution)
- Cassette agitation (0-500 RPM, 2 mm integrated orbital shaker)
Figure 1: Microreactor Prototype
The development phase of the project has been completed. During this phase the following techniques have been developed and refined:
- Fabrication and testing of cassettes with printed sensor dots and gas permeable membranes.
- Manufacture of a 24 reactor instrumentation block with integrated heaters, gas supply manifold, and pH/DO measurement system
- Validation and calibration of the system with buffers and model system
Significant data on the use of the instrument with several biological systems of interest to GtL has been collected using the microreactor system. We will present our results on the following:
- Fine tuning of parametric control using E. coli, B. subtilis and S. oneidensis.
- Demonstration of a parametric control experiment in an organism of DOE interest; The growth and the state of the metal reduction pathway in Shewanella oneidensis MR-1 is mapped as a function of pH, temperature, and dissolved oxygen.
- Determination of success or failure criteria on the basis of the sensor data and quality of replicates generated in the demonstration study.
Figure 2: Sample Data showing E. coli growth in LB with glucose. The lines marked N (orange) have neither pH control nor are supplied oxygen and thus become anaerobic and acidic. The lines marked A (blue) have oxygen control, but no pH control and thus remain aerobic and become basic. The lines marked P (green) are anaerobic, but have NH3 based pH control enabled with a setpoint of pH 7 and so maintain a constant pH.
* Presenting author
