Analysis of a Novel Immobilized Cell Reactor for Unstable Recombinant Cultures

本提案旨在开发一种固定化细胞反应器，通过选择性杀死没有重组DNA（没有质粒）的细胞来克服重组DNA不稳定性问题（质粒不稳定性）。这是用毒素（粘菌素）对大肠杆菌细胞进行的。一种质粒被用来赋予这些细胞对粘菌素的抗性，以及细胞表面对这种毒素的受体位点。粘菌素将被附着在一个由蔗糖珠组成的柱上，作为大肠杆菌的结合剂。那些没有质粒的细胞可能会被杀死并从柱上洗掉，而那些带有质粒的细胞可能会附着在柱上。上述系统对悬浮细胞培养（发酵）的优点是双重的。-毒素不在溶液中，不会被冲出反应器，从而节省了毒素的成本。-生物反应器的产物不需要经过净化步骤来消除毒素。PI被认为完全有资格进行拟议的研究，我建议为该提案提供资金。

This proposal seeks to develop an immobilized cell reactor in which a recombinant DNA instability problem (plasmid instability) is overcome by selectively killing cells without recombinant DNA (without a plasmid). This is to be done using a toxin (colicin) to E. coli cells. A plasmid is to be used that confers resistance of these cells to colicin as well as a receptor site on the cell surface to this toxin. Colicin is to be attached to a column of sepharose beads and act as a binding agent for the E. coli. Those cells without the plasmid presumably will be killed and wash off the column while those cells with the plasmid will presumably stay attached to the column. The advantage of the above system to a suspension cell culture (fermentation) is twofold. - The toxin is not in solution and will not wash out of the reactor thus saving on the cost of the toxin. - The product of the bioreactor will not need to pass through a purification step to eliminate the toxin. The PI is considered to be well qualified to carry out the proposed research and I recommend funding of this proposal.