Flow cytometry as a useful tool for process development: rapid evaluation of expression systems.
Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)(260)GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7-lac promoter and the P(BAD) promoter. The expression system based on the P(BAD) promoter was found to be superior to the T7-based system.
Patkar
Anant
A
Department of Chemical Engineering and Materials Science, University of Minnesota, 151 Amundson Hall, 421 Washington Avenue S.E., Minneapolis, MN 55455-0312, USA.
Vijayasankaran
Natarajan
N
Urry
Dan W
DW
Srienc
Friedrich
F
D001089:Arabinose / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000254:growth & development; D005434:Flow Cytometry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D005998:Glycine / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins; D007532:Isoleucine / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D011392:Proline / Q000096:biosynthesis / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D012107:Research Design; D012542:Scattering, Radiation; D014633:Valine / Q000096:biosynthesis / Q000235:genetics
D008164:Luminescent Proteins; D007532:Isoleucine; D049452:Green Fluorescent Proteins; D011392:Proline; D001089:Arabinose; D014633:Valine; D005998:Glycine