University of Natural Resources and Life Sciences, Vienna (BOKU) - Research portal
Gewählte Master / Diploma Thesis:
Eberhard Duerrschmid
(1996):
Induktion und Detektion des strigent response in Escherichia coli.
Master / Diploma Thesis,
BOKU-Universität für Bodenkultur.
UB BOKU
obvsg
- Abstract:
- Production of recombinant protein in E. coli puts a strong metabolic burden upon host cell metabolism. With the application of high expression rates , these loads lead to culture starvation within a single generation period due to consumption of monomers for replication of foreign DNA and for production of recombinant protein. To optimize recombinant product yields it is essential to characterize and measure this metabolic burden and to be able to adapt expression rates to the host cells metabolic capacity. The stringent control network provides a possibility to achieve these goals. By utilizing the signal molecule Guanosinetetraphosphate (ppGpp) the cell is able to adapt to C-source- or aminoacid-limitations by inhibition or at least downshift of highly energy-consuming processes e.g. protein or stable RNA synthesis. Because these reactions are also induced by endogenous limitations, ppGpp is an ideal indicator for the current metabolic state of the host cell during recombinant product formation. In this diploma thesis a procedure was developed to extract ppGpp and its precurors guanosine-di-and triphoshate as well as adenosine-di- and triphosphate from fermentation samples. Furthermore a Reversed Phase- Ionpair-HPLC method was established to quickly quantify these nucleotides. With these procedures a characterization of the stringent control network¿s behaviour to different limitations under fermentation conditions was achieved. Results show a similar level of ppGpp for exogenous and endogenous limitations.
- Beurteilende(r): Bayer Karl