Firebug: A Living Cell that Propagates Plasmids Built from an Artificial Genetic System

我们对生命的看法在很大程度上受到我们周围地球生命的影响。地球上所有的生命（我们所知道的）都来自一个共同的祖先，它们都具有基本相同的生物化学。这意味着我们对生物学的看法非常有偏见，只反映了我们对周围生物圈和我们自己的了解。这个项目的科学目标是扩展这一观点，这对于理解我们所知的生命现象和构成生命的分子之间的密切联系很重要。这种扩展的观点将是重要的，例如，如果我们在探索宇宙的过程中遇到外星生命。如果我们希望在商业、制造业和医药中使用类似生命的过程，这一点同样重要。这个项目面临的“巨大挑战”是创造一个能够使用完全合成形式的DNA的细胞；其中一个由6个核苷酸“字母”组成，比天然DNA中发现的核苷酸数量多两个。在追求这一挑战的过程中，我们将更多地了解新陈代谢、繁殖和基因调控的化学过程。除了作为能够进化的遗传系统的第二个例子的科学兴趣之外，细胞也将具有实用价值。该细胞将用于制造用于人类和动物诊断的分子，用于国土安全，以及作为生物安全平台的制造。本科生将参与研究活动，研究结果将继续为生命的起源和地外生命的性质提供有用的见解和观点。该项目由分子和细胞生物科学部的系统和合成生物学集群以及化学部门的生命过程化学共同资助。该项目将设计一种大肠杆菌菌株（“火虫”），它可以繁殖携带“第二代”人工扩展遗传信息系统（AEGIS）核苷酸的质粒DNA。在其最广泛的形式中，AEGIS将DNA中可独立复制的核苷酸的数量从4个增加到12个。该项目将首先在四种天然核苷酸（GACT）中添加两种名为（Z和P）的AEGIS组分，利用先前的工作(1)优化了第二代AEGIS组分的化学和酶促性能；(2)开发支持AEGIS DNA的体外分子生物学，包括六字母GACTZP PCR、测序和使用GACTZP DNA的内切酶、连接酶和激酶；(3)证明了GACTZP DNA的体外进化，包括在实验室中通过进化产生适配体；(4)工程激酶将dZ和dP转化为细胞内的三磷酸盐。为了通过合成生物学的下一个里程碑，使扩展的代码在细胞中发挥作用，将构建Firebug。这将涉及管理大肠杆菌容易出错的聚合酶和修复系统，将编码Z和P生物合成的酶移到染色体上，并将工程细胞置于选择状态下，以提高质粒携带的Z:P对复制的保真度。

Our view of life is very much influenced by the life on Earth around us. All of the life on Earth (that we know of) descends from a single common ancestor, and it all has fundamentally the same biochemistry. This means that our view of biology is very much biased, reflecting only what we know of our surrounding biosphere and ourselves. The scientific goal of this project is to expand this view, something important to understand the intimate connection between the phenomenon that we know as life and the molecules that underlie it. This expanded view will be important, for example, should we encounter alien life in our search of the cosmos. It is equally important if we wish to use life-like processes in commerce, manufacturing, and medicine. The "grand challenge" to be met by this project is to create a cell that is able to use an entirely synthetic form of DNA; one built from 6 nucleotide "letters", which is two more than the number of nucleotides found in natural DNA. In pursuing this challenge, more will be learned about the chemistry of metabolism, reproduction, and genetic regulation. In addition to its scientific interest as a second example of a genetic system able to evolve, the cell will also have practical value. This cell will be used to manufacture molecules for human and animal diagnostics, for homeland security, and for manufacturing as a bio safe platform. Undergraduate students will participate in the research activities, and the findings will continue to provide useful insights and perspectives on the origins of life and the nature of extraterrestrial life. This project is co-funded by the Systems and Synthetic Biology Cluster within the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes in the Chemistry Division.This project will engineer a strain of E. coli ("Firebug") that can propagate plasmid DNA carrying nucleotides from a "second-generation" artificially expanded genetic information system (AEGIS). In its broadest form, AEGIS increases the number of independently replicable nucleotides in DNA from 4 to 12. This project will begin by adding just two AEGIS components, named (Z and P), to the four natural nucleotides (GACT), exploiting prior work that has (1) optimized second-generation AEGIS components for their chemical and enzymatic performance; (2) developed in vitro molecular biology to support AEGIS DNA, including six-letter GACTZP PCR, sequencing, and endonucleases, ligases, and kinases that use GACTZP DNA; (3) demonstrated in vitro evolution of GACTZP DNA, including generation of aptamers by evolution in the laboratory; and (4) engineered kinases that convert dZ and dP to their triphosphates inside cells. To pass the next milestone in synthetic biology that makes the expanded code functional in the cell, Firebug will be constructed. This will involve managing E. coli's error-prone polymerases and repair systems, moving enzymes encoding biosynthesis of Z and P into the chromosome, and placing the engineered cells under selection to improve their fidelity of replication of plasmid-borne Z:P pairs.