50. J. Wu, L. Liu, T. Matsuda, Y. Zhao, A. Rebane, Mikhail Drobizhev, Y-F. Chang, S. Araki, Y. Arai, K. March, T. E. Hughes, K. Sagou, T. Miyata, T. Nagai*, W-h. Li*, R. E. Campbell*, “Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications”, ACS Chem. Neurosci., 2013. Accepted.
49. H. Hoi, T. Matsuda, T. Nagai, and R.E. Campbell*, “Highlightable Ca2+ indicators for live cell imaging”, J. Am. Chem. Soc., 2013, 135, 46-49.
48. S.C. Alford, J. Wu, Y. Zhao, R.E. Campbell, and T. Knöpfel*, “Optogenetic Reporters”. Biol. Cell, 2013, 105, 14-29.
47. A.L. McEvoy*, H. Hoi, M. Bates, E. Platonova, P.J. Cranfill, M.W. Davidson, H. Ewers, J. Liphardt, and R.E. Campbell*, “mMaple: a photoconvertible fluorescent protein for use in multiple imaging modalities”. PLoS ONE, 2012, 7(12): e51314.
46. S.C. Alford, Y. Ding, T. Simmen, and R.E. Campbell*, “Dimerization-Dependent Green and Yellow Fluorescent Proteins”. ACS Synth. Biol., 2012, 1, 569-575.
45. M. Funes-Huacca, A. Wu, E. Szepesvari, P. Rajendran, N. Kwan-Wong, A. Razgulin, Y. Shen, J. Kagira, R.E. Campbell and R. Derda*, “Portable self-contained cultures for phage and bacteria made of paper and tape”. Lab Chip, 2012, 12, 4269-4278.
43. R.E. Campbell*, “New Bioanalytical Tools and Devices: Chemistry leads the way”. Biotechnology Focus (Bioscienceworld), 2012, 16(4), 7-9.
42. K.D. Daze, T. Pinter, C.S. Beshara, A. Ibraheem, S.A. Minaker, M.C.F. Ma, R.J.M. Courtemanche, R.E. Campbell, and F. Hof*, “Supramolecular hosts that recognize methyllysines and disrupt the interaction between a modified histone tail and its epigenetic reader protein”. Chem. Sci., 2012, 3, 2695-2699.
41. S.C. Alford, A.S. Abdelfattah, Y. Ding, R.E. Campbell*, "A Fluorogenic Red Fluorescent Protein Heterodimer". Chem. Biol., 2012, 19, 353-360.
Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Forster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility.
To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library.
Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3x improved relative to that of the initially constructed biosensor (29%).
This special issue of Current Opinion in Chemical Biology dedicated to Molecular Imaging seeks not only to present a cross-sectional overview of recent technological advances in the field, but to highlight specific examples of how novel tools for visualizing real-time events in complex living systems has and continues to enable researchers to gain critical new insights into important and long-standing biological problems. This synergy between technology and application continues to push the boundaries of how we understand the natural world around us and further drives the need for innovation in the development of new Molecular Imaging tools.