Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor.
Synthesis of an operon fusion protein was investigated in batch and fed-batch cultures at high cell densities of recombinant Escherichia coli JM105 [pBAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L(-1) DCW) to be attained. This was believed to be the highest reported value for dry cell mass of E. coli strain JM105 expressing two recombinant proteins. Transcription of the two reporter genes, green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT), was under the control of the p(BAD) promoter of the araBAD (arabinose) operon. Each protein was independently translated via separate ribosome binding sites. CAT served as a model recombinant protein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monitored on-line using an intensity-based optical sensor. A linear correlation between the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product under conditions of high biomass concentration and high productivity.
DeLisa
M P
MP
Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland 20742, USA.
Li
J
J
Rao
G
G
Weigand
W A
WA
Bentley
W E
WE
D001089:Arabinose / Q000494:pharmacology; D015374:Biosensing Techniques; D001709:Biotechnology; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D015169:Colony Count, Microbial; D003470:Culture Media; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005285:Fermentation; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009862:Online Systems; D009876:Operon; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics*
D003470:Culture Media; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001089:Arabinose; D015500:Chloramphenicol O-Acetyltransferase