Continuous Production of RNA Using a Stir-Cell Bioreactor.
Abstract
The increased study of ribonucleic acids (RNAs) due to the discovery of their potential catalytic and selective binding properties has prompted research into developing a large-scale, cost-effective RNA production strategy. Unfortunately large amounts of RNA, made either by direct chemical or enzymatic synthesis, are expensive and difficult to produce because of the high material cost and limitations found in batch mode processes. In an effort to make large amounts of RNA efficiently and effectively, a process using immobilized DNA templates and bacteriophage T7 polymerase in a continuous stir-cell membrane bioreactor has been investigated. This method decreases the overall cost of the process by retaining the polymerase and template while separating the newly formed RNA using an ultrafiltration membrane. This membrane has a molecular weight cut-off of 100,000 daltons and is a low protein binding, hydrophilic, regenerated cellulosic disk. Membrane studies indicated that the transmission of RNA is affected by the transcription mixture. Further studies of the stir-cell bioreactor indicated that the polymerase is possibly denatured causing the transcription reaction to plateau. The continuous addition of precursor nucleotide triphosphates and buffer does not prevent the reaction from stopping, but the addition of polymerase causes in increase in RNA production. Optimized NTP concentrations produce more RNA with less abortive transcripts compared to the products of reactions with limiting NTPs. Continuous production of RNA is a viable alternative for its large scale production Reactions have been performed with a continuous feed stream of NTPs and buffer for up to 300 minutes.
Document Details
- Document Type
- Technical Report
- Publication Date
- Mar 01, 1995
- Accession Number
- ADA292412
Entities
People
- Neal T. Williams
Organizations
- Air Force Institute of Technology