IP-Free™ Protein Paintbox™

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Raul Duran, Matthew Jones, Claes Gustaffson, Sridhar Govindarajan, Jon Ness,Drew Regitsky, Drew Endy, Jerome Bonnet, Medini Gore, Laura Whitman, Jeremy Minshull

DNA 2.0, United States

Fluorescent proteins are a valuable tool for research, with uses ranging from reporters in transgenic systems to biosensors. The tangle of intellectual property restrictions, as well as protein characteristics like brightness, oligomeric state and sensitivity to environmental conditions limits the breadth of tasks to which fluorescent proteins can be applied. We have created a large set of new colored and fluorescent proteins by backtranslating a set of sequences from Genbank, using GeneDesigner software to simultaneously minimize sequence differences and match an E coli codon bias. Oligonucleotides designed to synthesize each gene were combined to create chimeric genes. The resultant chimeras were selected under various illuminations. Analysis of sequence and spectral data allowed us to infer correlations between them. Correlations were then tested by creating specific individual sequences and determining whether their spectral properties were as predicted. Three amino acid positions were found to be individually responsible for cyan, green or yellow fluorescence emission. We also identified variants with non-overlapping spectra as candidates for applications using more than one fluorescent protein, for example microscopy and FRET (Fluorescence Resonance Energy transfer) applications. DNA2.0 will make its Protein Paintbox™ available without intellectual property restrictions, for the Synthetic Biology research community to improve upon and to incorporate into innovative products.