In vitro Genetic Analysis for Metabolic Engineering of Cell-Free Protein Synthesis

该项目的长期目标是开发有效和灵敏的基因组代谢调查方法。该项目将尝试利用基因组序列的可用性，大规模并行分子生物学技术，以及高效的无细胞蛋白质合成，以获得基因组中所有可溶性酶的功能表达，每个反应室一种蛋白质。 通过这种方法，几乎每一种酶都可以快速测试其对目标代谢过程的影响。这种能力将通过提高无细胞蛋白质合成的性能来证明。 这个项目将试图表明，无细胞条件下可以开发模拟E。大肠杆菌细胞质，并提供各种辅基，使复杂的，多聚体蛋白质将正确折叠，成为生物活性。 该项目还试图表明，单一的生物活性蛋白质可以对无细胞蛋白质合成产生深远的影响，并且这些影响将基于蛋白质的已知细胞内功能进行预测。已经选择了十种蛋白质来代表蛋白质表达和活化中的各种挑战。 如果这个项目成功，定向基因改变可以以更高的概率实现预期的结果。

The long term goal of this project is to develop methods for efficient and sensitive metabolic surveys of genomes. This project will attempt to use the availability of genomic sequences, massively parallelmolecular biology techniques, and efficient cell-free protein synthesis to obtain functional expression of all the soluble enzymes in a genome, one protein per reaction chamber. With this approach, nearly every enzyme could be tested rapidly for its effect on a targeted metabolic process. This capability would be demonstrated by improving the performance of cell-free protein synthesis. This project will attempt to show that cell-free conditions can be developed to mimic the E. coli cytoplasm and to provide a variety of prosthetic groups so that complex, multimeric proteins will fold properly to become bioactive. This project would also attempt to show that single bioactive proteins can have a profound effect on cell-free protein synthesis and that these effects would be predicted based on the known, intracellular function of the protein. Ten proteins have been chosen to represent various challenges in protein expression and activation. If this project is successful, directed genetic changes could be made with a much higher probability of the desired outcome.