Periodic Reporting for period 1 - AnBioLED (AnBioLED Ancestral Fluorescent Proteins as Biophosphors for Light-Emitting Diodes)
Reporting period: 2023-01-01 to 2024-12-31
Sustainable light technologies are overcoming certain challenges in order to become attractive enough to compete with market options. One of such challenges is the replacement of toxic or rear-Earth materials like cadmium or cerium as color downconverters in Light Emitting Diodes (LEDs). The usage of Fluorescent Proteins (FPs) is a novel sustainable approach, leading to the development of Bio-Hybrid LEDs (Bio-HLEDs). However, a second challenge to overcome is the device stability in Bio-HLEDs. Since FPs are not biologically adapted to be used as a technological component in light devices, the project AnBioLED (Ancestral fluorescent proteins as Biophosphors for Light-Emitting Diodes) aimed to estimate ancestral-like FPs out of the sequences of present day FPs. This was made under the premise that ancestral sequence reconstruction tends to give place to proteins that are more resistant to certain stresses than present day FPs. During the AnBioLED project, ancestral-like FPs with exceptionally efficient color emission and better stability than previous FPs used so far in Bio-HLEDs have been discovered. The expected impacts of this project are: 1) the development of LEDs with biodegradable ancestral-like FPs as color downconverters that can compete with previous state-of-the-art in terms of color emission and stability and 2) the usage of ancestral-like FPs as scaffolds to engineer a novel family of proteins for technological applications.
1.- Ancestral-like FPs design: bioinformatics protocols to reconstruct and optimize genetic sequences of ancestral-like proteins with the excellent photoluminescence features of representative present day FPs. This step focused on the collection of 22 sequences of present day FPs with specific attributes like color emission, high quantum yield, etc; which later on allowed the expansion of the search for more sequences like them, leading to a finals data set of 221 present day FPs. Using this data set, multiple sequence alignments and ancestral sequence reconstruction were applied. In order to have an estimate of how likely the estimated ancestral sequences were going to fold into a functional protein, further computational analyses were developed, which were successfully applied to other proteins and culminated in publications, along with methodologies to assess interaction with additives, chromophore geometries and circularly polarized light emission.
2.- Ancestral-like FPs validation: synthetic biology for the optimization of the expression/purification of the estimated ancestral-like FPs, and their photophysical-thermal-mechanical characterization on FP-polymer composites. During this step, plasmid vectors harboring the ancestral-like sequences were carefully designed for further bacterial expression. The pure proteins were characterized both in solution and solid (once in the polymer composites) for their photo-physical properties
3.- Ancestral FPs Bio-HLEDs: optoelectronics for the fabrication- characterization of Bio-HLEDs. In this final step, the materials were used as downconverters on LEDs, and the several features were monitored during operation, such as temperature, color emission and stability.
2.- Ancestral-like FPs validation: synthetic biology for the optimization of the expression/purification of the estimated ancestral-like FPs, and their photophysical-thermal-mechanical characterization on FP-polymer composites. During this step, plasmid vectors harboring the ancestral-like sequences were carefully designed for further bacterial expression. The pure proteins were characterized both in solution and solid (once in the polymer composites) for their photo-physical properties
3.- Ancestral FPs Bio-HLEDs: optoelectronics for the fabrication- characterization of Bio-HLEDs. In this final step, the materials were used as downconverters on LEDs, and the several features were monitored during operation, such as temperature, color emission and stability.
One of the ancestral-like FPs has a green emission and an exceptionally high quantum yield in solution of 80%, which is rare in present day FPs. Even after such value decreased to 76% when the protein was mixed with the polymer composite, such value is still remarkable. Interestingly, the ancestral-like protein has a double value, as it can convert into a red emissive protein upon a stimulus, which is a feature only a few present day FPs have. Even in the red form, the protein still has outstanding quantum yields of 80% in solution and 54% in solid, but the most interesting feature is that, in terms of stability, this ancestral-like FP outperforms any other FP used in the same configuration of device, with even 2-fold with respect to the most comparable FP. These results have an impact in further development for more stable Bio-HLEDs devices, as they can be further engineered for achieving different colors, photoswitching, improvement upon certain additives, chemical modifications, etc.