Rapid determination of plasmid copy number.
Plasmid copy number, the number of expression vectors per host cell, is a key variable in recombinant microbial cultivation. Therefore, it would be very helpful, if the plasmid copy number could be determined during the operating process period. A rapid quantification of this important process variable would even open the possibility of its use in process control. However current assays like gel electrophoresis, CsCl-gradient centrifugation, HPLC and other methods are time consuming and difficult to quantify. Indirect methods, like the correlation of copy number with, e.g. the activity of an enzyme, coded on the plasmid, are prone to errors due to the production kinetics, turnover rate and protein denaturation. Here, a method is presented, which enables the plasmid copy number to be determined in less than 30 min. This novel procedure based on plasmid isolation by means of a commercial DNA-isolation kit and quantification by capillary electrophoresis, should allow the copy number to be used in process control.
Schmidt
T
T
Lehrstuhl für Fermentationstechnik, Technische Fakultat, Universität Bielefeld, Germany.
Friehs
K
K
Flaschel
E
E
D019075:Electrophoresis, Capillary / Q000379:methods*; D005787:Gene Frequency; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic
D011933:Reagent Kits, Diagnostic