Several species have had their genomes sequenced. The focus is now shifting to a thorough study of what these sequences code for by examining the resulting proteins. Accounting for millions of proteins expressed by thousands of genes is going to require a combinatorial effort. Cells will have to cultured in controlled environments and exposed to a variety of environmental and physiological conditions in order to elicit differential responses so that the factors responsible for gene expression can be elucidated. A major limitation is the ability to grow cells under controlled conditions in large numbers such that thousands of experiments may be performed. The current state-of-the-art requires the use of instrumented bioreactors that are bulky and expensive and not amenable to being run in large numbers in parallel. Whereas microtiter plates do offer the ability for large numbers of experiments, they lack the sensors and instrumentation to enable environmental control. We are proposing to bridge this gap by utilizing low-cost, non-invasive optical sensor technology in order to make controlled cell cultivation possible in a system analogous to microtiter plates. Our goal is to make the monitoring and control of pO2, pH, and Optical Density (to measure cell growth) facile. Our approach is to use miniature scaled down versions (called microbioreactors) of liter scale bioreactors. We employ optical sensing techniques where an analyte-sensitive patch is placed in each microbioreactor where cells are grown and monitored optically from outside. In addition, we will equip the instrumentation for monitoring Green Fluorescent Protein (GFP), as this is a widely used marker of gene expression. The proposed system will allow 24 milliliter scale bioreactor experiments to be simultaneously conducted under precisely controlled conditions at about the same cost as a single laboratory bioreactor. Specifically in the first year (R21 period), we will: 1. Construct a cell cultivation platform consisting of 24 microbioreactors to be used with an instrument platform and equipped with sensors to monitor pO2, pH, Optical Density and GFP. 2. Demonstrate its use for simultaneous monitoring of 24 E. coli fermentations for the four parameters. These will also serve as our milestones for continuation onto the next phase. Over the next three years (R33 period), we will 3. Implement strategies for control of pO2, pH and timed nutrient addition. 4. Validate the system with a study of oxidative stress responses in E. coli using GFP as the reporter gene and demonstrate feasibility for mammalian cell culture. The above technology has the potential to significantly impact practices ranging from basic studies of gene expression to bioprocess development as pertaining to the development of pharmaceuticals and vaccines.
| Kostov, Yordan; Smith, Derek S; Tolosa, Leah et al. (2005) Directional surface plasmon-coupled emission from a 3 nm green fluorescent protein monolayer. Biotechnol Prog 21:1731-5 |
