Engineering the central dogma using emulsion based directed evolution
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The central dogma of molecular biology forms the most basic (and fundamental) paradigm of how life operates. Despite its elegant simplicity, scientists are still uncovering enigmas of the central dogma - which has been shaped throughout billions of years of the Darwinian process. Even though the core concepts of the central dogma have largely been untouched by evolution (the universality of the genetic code, amino acid utilization, DNA/RNA base identity) scientific advances have shown that these fundamental properties can be altered dramatically. This implies the architectures of life are pliable and likely the result of extreme optimization and fine tuning of semi-random events that took place soon after the origin of life. Reengineering the parameters of life offers a unique way of testing evolutionary processes and perceived optimality of its components. Naturally, coaxing proteins and nucleic acids to function in an unnatural fashion is difficult. Development of techniques to enable these changes has relied heavily on the exploitation of water-in-oil emulsions (or, in vitro compartmentalization), which allows directed evolution at the single cell or even single molecule level. In particular, compartmentalized partnered replication (CPR) is a dual mode selection technique, coupling the in vivo functionality of a gene with the in vitro amplification via emulsion PCR. The CPR technique has enabled the development of synthetic promoter recognition by T7 RNA polymerase, unnatural amino acid incorporation by aminoacyl tRNA synthetase engineering, genetic code reassignment through tRNA evolution, and transcriptional regulation using repressors with novel allosteric effector molecules and operator binding sites. Using a similar technique, the template recognition of an Archaeal DNA polymerase was altered such that the polymerase utilizes both DNA and RNA templates with similar efficiencies. This resulted in a reverse transcriptase that can functionally proofread on RNA templates. These technologies will continue to play a pivotal role in the future development of particular aspects of the central dogma. As certain steps in this process are tweaked to have alternative functionalities and combined together, the gap between natural life and synthetically modified life widens and gives the Darwinian process of evolution new areas to explore.