The general goal of this proposal is to assemble directed mutant gene sets quickly, cheaply, easily, and reliably. Mutant gene sets can share sub-assemblies, yielding savings of time, cost, and effort not possible if designed and assembled all as single genes. The approach is to build upon experience gained in Phase I, to extend and generalize methods of DNA design and assembly, to carry out work plans for each major subsystem, and to validate the resulting system in the wet-lab. Innovation lies in applying computational optimization to mutant sequence assembly, in bringing cluster computing and thermodynamics to the wet-lab, in the integration of different methods, and in the general approach taken. The significance is a method for rapidly, inexpensively, and efficiently constructing mutant gene sets of biomedical importance. We will provide the scientific and medical communities with the ability to perform rapid, inexpensive, and accurate mutational studies of any protein(s), using a scaffold based on synthetic genes computationally optimized for DNA self-assembly and for expression and translation in an in vitro or in vivo system of choice. This capability will be immediately and directly useful to biomedical researchers, and serve as a platform for protein and metabolic engineering. Mutational studies are one of the primary methods used by biomedical researchers to study and modify protein structure and function. Mutations of a parental gene are the main route to improved protein expression and function in biopharmaceuticals and industrial enzymes. Function follows sequence. For many reasons, mutant gene sets are important life science research tools. The computational goal of the project is to demonstrate that directed yet complex libraries can be designed and built using this technology. The biological goals have been chosen for their biomedical significance, because integrase is an important retroviral drug target and its understanding is a major medical goal: (1) to find active soluble integrases (IN) for HIV and Ty3 that might lead to crystal structure(s) of active IN(s);(2) to understand the structural basis of retroviral integration and position specific retrovirus- like integration;and (3) to derive chimeric proteins for position-specific retrovirus vectors, as the position-specific insertion of Ty3 IN might lead to better gene therapy vectors.
The general goal of this proposal is to provide the scientific and medical communities with directed mutant gene sets quickly, cheaply, easily, and reliably. Mutational studies are one of the primary methods used by biomedical researchers to study and modify protein structure and function. Mutations of a parental gene are the main route to improved protein expression and function in biopharmaceuticals and industrial enzymes.
| Wassman, Christopher D; Baronio, Roberta; Demir, Özlem et al. (2013) Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53. Nat Commun 4:1407 |
