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. Author manuscript; available in PMC: 2014 Apr 21.
Published in final edited form as: Chem Soc Rev. 2013 Jan 29;42(8):3441–3452. doi: 10.1039/c3cs35458j

Fig. 5.

Fig. 5

Molecular evolution steps, methods and techniques, and specific conditions in the course of development of the BphP-based NIR fluorescent proteins, biosensors, and optogenetic tools. Vertical arrows indicate the typical order of the evolution steps such as gene construction and mutagenesis, biological hosts for protein expression, instrumental methods of screening, protein characterization in vitro and in cells. Methods and techniques proposed for each molecular evolution step are subdivided per the proposed NIR probes. Specific conditions indicate particular qualities of BphPs that should be considered for each directed evolution step. HTS is a high-throughput screening, FACS is a fluorescence-activated cell sorter, and λ is a wavelength. See also Table 2 for details on knowledge-based mutagenesis.