Genetic approaches to the detection of contaminants in Escherichia coli fermentations.
In facilities which cultivate more than one rDNA organism, contamination by the same species is difficult to detect. Since the same species may be producing a different product in an adjacent fermentor or in the same vessel in a subsequent procedure, the possibility of cross-product contamination must be considered. Here we describe a simple, sensitive, and reliable technique for the detection of same-species contamination. The assay uses negative genetic markers such as the inability to use a carbohydrate, e.g., ribose. When the facility is managed to use the ribose marker for only one product, this culture can be plated on ribose minimal medium to allow rapid and sensitive detection of contaminants. If the facility is used for several rDNA products, multiple carbohydrate markers per strain can be used so that a limited number of markers can differentiate among larger host collections. The approach was developed and tested using Escherichia coli as the host organism. If hosts with auxotrophies, e.g., for amino acids, are used in the facility, the plate medium can be supplemented. When this technique is combined with existing methods for detecting different species and bacteriophage contamination, all three classes of biological contamination can be detected.
Swartz
J R
JR
Department of Cell Culture, Genentech, Inc., South San Francisco, California 94080, USA. swartz.jim@gene.com
McFarland
N
N
D004269:DNA, Bacterial / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D004866:Equipment Contamination; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005240:Feasibility Studies; D005285:Fermentation; D005819:Genetic Markers; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005819:Genetic Markers