Assessment of physiological conditions in E. coli fermentations by epifluorescent microscopy and image analysis.
The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes.
Carneiro
Sónia
S
Centre of Biological Engineering, Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Amaral
António L
AL
Veloso
Ana C A
AC
Dias
Teresa
T
Peres
António M
AM
Ferreira
Eugénio C
EC
Rocha
Isabel
I
D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability; D008856:Microscopy, Fluorescence / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins