In situ measurement of bioluminescence and fluorescence in an integrated microbioreactor.
Reporter strains of bacteria that emit light or a fluorescent marker in response to specific conditions in their environment are having a significant impact in many areas of biology, including toxicity assays for environmental pollutants, chemical detection, and gene expression profiling. We have demonstrated methods for in situ measurements of bioluminescence and fluorescence from bacterial cultures grown in 50 microL instrumented microbioreactors. Results from microbioreactors were compared to results obtained from conventional 500 mL batch bioreactors and shake flasks. Experiments were conducted with reporter strains of Escherichia coli in which luxCDABE or gfp was fused to a promoter that was either expressed constitutively, or that responded to oxygen limitation. With these reporter strains, we have demonstrated the ability to obtain information on growth conditions within the microbioreactor. We have also shown that the large aspect ratio of the microbioreactor provides a unique advantage over measurements in larger bioreactors by reducing the inner filter effect in on-line measurements and eliminating the need for error-prone off-line dilutions. In addition, continuous on-line monitoring of genes in real-time, when expanded to include entire reporter libraries, could potentially provide a true dynamic picture of cellular gene expression from which the kinetics of gene expression can be untangled and elucidated.
Zanzotto
Andrea
A
Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, USA.
Boccazzi
Paolo
P
Gorret
Nathalie
N
Van Dyk
Tina K
TK
Sinskey
Anthony J
AJ
Jensen
Klavs F
KF
D019149:Bioreactors; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins