GCE4All projects work through a repetitive process of development and beta-testing which culminates in the release of GCE technology and tools to the broader community.

Our work begins with Technology Optimization Projects (TOPs), the core of our research activities which serve as the foundation for all other GCE4All activities. In each optimization project, a Center research team optimizes (and even creates when necessary) select critically-needed GCE technologies, bringing them to a stage where they are robust and ready to be disseminated to the broader research community, experts and non-experts alike … i.e. GCE for all!

During this process, Driving Biomedical Projects (DBPs) act as "beta-testers" of developing technologies. Researchers in DBP labs will apply the technologies to their specific research projects. The center then works with their feedback, allowing the technology to be refined until it meets their requirements and is successfully implemented to achieve research breakthroughs.

After successful implementation by our DBPs, the Community Engagement arm of the Center begins the process of making the technology sustainably available to the broader community through non-Center entities such as repositories or companies. While this sustainable distribution is being put into place, direct distribution in a limited fashion may be provided by the Center to some "early adopters" who ask us for access to the technology as soon as it has been proven reliable.

Technology Optimization Projects

Each TOP is focused on optimizing a subset of GCE tools that can have great impact in advancing biomedical research, but need to be made more robust and efficient in terms of the incorporation of certain types of non-canonical amino acids (ncAAs) and in terms of protocols for optimally handling and using the GCE-produced designer proteins.

The advances in Genetic Code Expansion technology targeted by GCE4All are organized into two TOPs:

• TOP-1 focuses on ncAAs for bioorthogonal ligations
• TOP-2 focuses on ncAAs that can serve as biochemical probes as well as ncAAs that represent post-translational modifications

As the TOPs develop technologies, GCE4All investigators will gain valuable experience regarding how to most effectively develop, optimize and implement GCE tools for any given ncAA. As a synergistic outcome, the combined experience of the TDPs will be leveraged to develop robust approaches and protocols for GCE tool development. We call these “GCE bridges,” and our vision is that these bridges will give any molecular biology lab the ability to develop – from scratch – a full set of efficient GCE tools for a novel ncAA of interest. The four GCE bridges to be built are:

• The bridge to access a new ncAA through developing the needed RS/tRNA pair
• Given an RS/tRNA pair, the bridge to optimize ncAA-protein expression in E. coli
• Given an RS/tRNA pair, the bridge to optimize ncAA-protein expression in mammalian cells
• Given an RS/tRNA pair with optimized expression, the bridge to create a stable GCE mammalian cell line

TOP-1: GCE Technologies for Bioorthogonal ligations

Current TOP-1 projects focus on incorporating tetrazine amino acids into proteins and reacting them with trans-cyclooctane (TCO) reactive groups that can be attached to a fluorescent dye or via a linker to another protein or surface (building on the chemistry reported by Blizzard et al 2015). GCE technologies enabling efficient tetrazine amino acid incorporation into proteins in both E. coli and mammalian expression hosts will be optimized alongside conditions for high-yield reactions with TCO-activated ligation targets. When expressed in mammalian cells, Tet-proteins react intracellularly providing unprecedented abilities to modify and study proteins in their physiological settings. Once optimized, GCE bioorthogonal ligations will enable attachment of larger fluorophores further enabling the addition of polymers, drugs, imaging agents, or other biomolecules and materials to ultimately impact a wide range of science.

TOP-2: GCE Technologies for Biochemical Probes and Post-translational modifications (PTMs)

Current TOP2 projects focus on optimizing GCE technologies to incorporate ncAAs that are: (1) highly fluorescent probes, such as acridone (Acd); (2) fluorine containing amino acids (e.g. F_x-Phe) useful for probing pi-cation interactions and for nuclear magnetic resonance characterization of protein structure and dynamics; and (3) the PTMs phospho-serine (pSer), phospho-threonine (pThr) and a non-hydrolyzable mimic of pSer (nhpSer). These incorporations are taking place in E. coli or in mammalian cells, and in some cases in both. When optimized, GCE technologies for biochemical and post-translational modifications will be useful in gaining greater understanding of protein regulation and interactions, studying oxidative stress and its relationship to disease, uncovering mechanisms of molecular recognition including ion channel selectivity, and revealing protein dynamics and conformational changes.

PermaPhos: Revealing New Functions of Phosphorylated Proteins (Seminar Recording)