座谈会活动方案:《科学家》25周年特刊：生物合成－修补生命

20世纪90年代晚期，一些物理学家和工程师对生物学产生了极大的兴趣。人类基因组计划实现了越来越多的基因测序：描绘了细胞蛋白质构件蓝图，获取了有关生命体分子机械的大量信息。但问题在于，没有足够多的生物学家在研究如何让这些不同的基因和蛋白质协同作用产生活的有机体。

It was around this time that Boston University bioengineer James Collins saw his chance to inject a little engineering know-how into the study of biology. There were two ways to go about it, he figured—either disassemble cells or build them. “A burgeoning young engineer [is] either the kind of kid who takes stuff apart to try to figure out how it works, or [he’s] the kid who puts stuff together,” Collins says. Though both approaches seemed promising, there simply wasn’t enough known about the structures or functions of the genes and their protein products to infer how all the parts worked together by taking a cell apart, piece by piece.

就在这个时期，波士顿大学的生物工程师James Collins看到了机会，他把一些工程学的技术知识运用到生物学的研究当中。他说，有两种办法可以实现这种融合：一种就是将细胞拆解，另一种就是细胞组合。“一个年轻的工程师或许会像孩子一般把东西拆开，然后研究它的内部原理，或者他本身就是个孩子，把不同的东西结合起来，”Collins说道。尽管两种方式看起来都不错，但是因为人们对于基因的结构与功能和它们的蛋白质物质还知之甚少，所以通过一片片的分子，还很难推断出这些物质是如何工作的。

“Reverse engineering seems to be too challenging,” Collins recalls musing to his then grad student Tim Gardner. “But can we do forward engineering? Can we take parts from cells and put them together in circuits, just as an electrical engineer might?”

”逆转操作似乎很有挑战性，“Collins回忆他当时若有所思地对他后来的研究生Tim Gardner说道。“但我们是否可以实现正向操作呢？我们是否可以从细胞中提取片段然后像电力工程师一样把他们连成电路呢？”

The answer was yes. After two years of tweaking various characteristics of transcriptional repressors in E. coli, the team succeeded in constructing biology’s first synthetic toggle switch—two repressor genes controlled by two promoters that caused their respective repressors to be expressed by default. The repressors were designed to inactivate each other, however, such that the two genes would never be fully expressed at the same time. The addition of a stimulus, such as a chemical pulse to suppress one gene long enough for the other to come on, allowed the system to flip from one stable state (gene A on, gene B off) to its other stable state (A off, B on).

答案是肯定的。通过两年对大肠杆菌的转录阻遏物不同特征的研究，该团队成功地造出了生物第一个合成基因开关－两个阻遏物基因由两个启动子控制，使得相关的阻遏物通过默认方式被表达。每个阻遏物相对于另一个都失去活性，然而，两个基因永远不可能同时得到完全表达。除刺激物外，长时间抑制一个基因以等待另一个基因开启的化学脉冲，使得系统可以从一个稳定的阶段（基因A启动，基因B关闭）到一个稳定的阶段（A关闭，B开启）。

The results were published in 2000, alongside a paper from physicist Stanislas Leibler’s lab at Princeton University, which had undertaken a similar, but independent, project. Much like Collins with Gardner, Leibler teamed up with his graduate student Michael Elowitz to build an oscillator, which, like Collins’s toggle switches, used transcriptional repressors in E. coli. The Princeton team engineered three genes to inhibit each other in a cyclical manner, rock-paper-scissors style, with each gene repressing the next when a threshold concentration of its gene product had been reached. The result was the periodic of all three genes—monitored by the periodic glow of green fluorescent protein (GFP), whose was linked to another copy of a promoter controlling one of the three repressors.

这些结果在2000年得以公布，普林斯顿大学物理学家Stanislas Leibler的实验室同时发表了一篇论文，他也进行了类似独立的项目。像Coolions 带着 Gardner做项目一样,Leibler和他的研究生Michael Elowitz 制造了一个振荡器，也是像Collion的基因开关一样，运用大肠杆菌的转录阻遏物。普林斯顿团队让三个基因以一种循环的，“石头，剪子，布”的方式相互抑制，也就是让每个基因在基因物质达到临界浓度的时候后对下一个基因进行遏制。这样做就实现了全部三个基因的周期性表达－这个过程由绿色荧光蛋白（GFP）的周期性闪烁来监控，这个表达与一个启动子的另一个副本相连，这个启动子控制着三个阻遏物质当中的一个。

The two publications are now widely cited as the seminal papers of synthetic biology, though neither paper received much publicity at the time. “[We were] kind of a ragtag group of engineers and physicists who were essentially amateurs in molecular biology,” Collins says. But in the last decade, many trained molecular and cell biologists have turned to syn bio, designing synthetic circuits built from biological components and branching out from the transcriptional regulation tools of Leibler, now at Rockefeller University, and Collins to add translation and post-translation components.

这两篇论文如今已经称为合成生物学的研讨篇目，尽管在当时，任何一篇文章都没有引起公众注意。“我们只是一群混杂的工程师和物理学家，在分子生物学领域都很业务。”Collins说。但是在过去的十年，很多经过训练的分子和细胞生物学家也都转向了合成生物领域，通过对Leibler常规转录工具运用的扩展，来设计合成电路, 用Collins的工具来增加转译和后转译部分。

The methods for actually manufacturing the circuits have also improved. While the Collins and Leibler teams were essentially cutting and pasting existing genes, J. Craig Venter and his colleagues went for a ground-up approach. They took the blueprint of a known bacterial genome and rebuilt the entire sequence, stitching together genes chemically manufactured by an automated DNA synthesizer. The genome was then inserted into the nucleus of another bacterium, with May 2010 headlines announcing the creation of the first cell to run on a genome synthesized entirely from scratch. 

Many researchers still use the basic cut-and-paste approach, however, employing well-vetted and still advancing genome-editing technologies to select different bits of DNA, called BioBricks, from living organisms and piece them together to form novel circuits. Others, like George Church of Harvard University, fall somewhere in the middle, synthesizing individual genetic components using oligonucleotide chips, then piecing them together. “I think it’s an open question as to whether the core of synthetic biology is going to make things by BioBricks, by total synthesis, or from scratch from chips in a modular way,” says Church.

很多研究者仍旧运用复制粘贴的方式，然而，他们是用已经修正并持续发展的基因编辑技术来选择来自生命有机体的不同的DNA，并把它们连接起来形成新的电路。其他一些人，比如哈佛大学的George Church, 采取比较中间的位置，运用寡核苷酸芯片来合成个体基因成分，然后把它们拼在一起。“关于和成生物学的核心到底是用生物砖，全部合成，还是从零做起，以分子的方式从芯片做起，是一个可以继续探讨的问题，”Church说。

Regardless of how the circuits are assembled, engineered organisms hold potential in a wide range of fields, including biofuel production, agricultural innovation, and biomedical advances. One of the most successful medical applications has been the engineering of yeast to produce a precursor of the antimalarial drug artemisinin, a natural product of the plant Artemesia annua. The production of the drug is currently limited to small farms in Southeast Asia, where farmers grow the plants and extract the drug using relatively crude techniques, making the drug expensive and often in short supply—a bad combination for the developing nations that need it most.

无论电路是如何组成的，设计出的有机体在很多领域都具有潜力，包括生物染料生产，农业创新，生物医药进步。比较成功的医疗应用之一是制成酵母来生产抗虐药物黄蒿素前导－黄花蒿的自然物质。这种药物目前只在东南亚的一些小农产生产，它很昂贵并且经常供不应求－发展中国家最需要这种药物，所以这种情况很糟糕。

To address these problems, Jay Keasling of the Lawrence Berkeley National Laboratory and his colleagues decided to rebuild the artemisinin pathway in a more manageable microbial system. After several years of tweaking the molecular components first in E. coli, then in yeast, the researchers succeeded in building a synthetic circuit in yeast cells that generates a healthy supply of artemisinic acid—an artemisinin precursor. “If you were to take something like a 100,000-liter fermenter, and grow up our artemisinin-producing yeast, running that full time you could probably get enough artemisinin for the entire world,” Keasling says. With funding from the Bill & Melinda Gates Foundation and partnerships with California-based biotech Amyris, the Institute for OneWorld Health, and pharmaceutical giant Sanofi to optimize and scale up production and distribute the product to Africa, Keasling and his colleagues expect that the yeast-derived artemisinin will be commercially available by the end of this year, and that drugs containing the product will hit the market in early 2012.

为了解决这些问题，劳伦斯伯克利国家实验室的Jay Keasling和他的同事决定以更加可管理的微生物体系去重新重建青蒿素路径。通过组合大肠杆菌和酵母的分子，研究者成功地在酵母细胞中形成整合电路，形成青蒿素酸的健康供应－青蒿酸前驱。“如果你用大约10万升的发酵槽，来生产青蒿素产生的酵母，一直运作下去就可以为全世界生产出足够的青蒿素，”Keasling说道。通过比尔与美琳达盖茨基金会和与加州理工大学的伙伴关系，美国人类健康研究所和医药巨头赛诺菲来最优化和拓展生产，把产品发放到非洲，Keasling和他的同事希望由酵母产生的青蒿素将会于今年低开始用于商业用途，含有这种产品的药物会在2012年初在市场问世。

Another synthetic biology inspired malaria project aims to stop transmission of the disease at the level of its vectors by engineering a genetic system to establish itself in a mosquito population. While researchers have successfully engineered mosquitos to be resistant to infection by the malaria parasite, introducing those mosquitos into the wild is not likely to result in sufficient spread of the resistance, as the wild-type genes will vastly outnumber the introduced variety. Something, such as a significant fitness advantage, must help drive the new genes into the population. Geneticist Bruce Hay and his team at Caltech got their inspiration for a solution to this problem from the Medea toxin/antidote genetic element in Tribolium beetles, in which a toxic maternal gene product kills any embryos that do not inherit the element, ensuring its quick spread through the population. Armed with 50+ years of Drosophila genetics knowledge, the researchers created a genetic element, Medeamyd88-1, which caused mother flies to produce eggs that only survived if they received a copy of the element.

In laboratory tests, Medeamyd88-1 quickly spread through the population, such that every individual carried the element by the 12th generation. Hay’s group is now working on developing a similar system in disease-carrying mosquitos. If he succeeds, “then it becomes a question of can we link these two pieces of biology together,” Hay says—the gene that makes the mosquitos disease-resistant and the Medea element that drives it through the population.

As was the intention of some of the field’s founding engineers, synthetic biology also promises to help researchers understand the basic rules of cellular function in ways that traditional biology hasn’t been able to, says Elowitz, now a professor at Caltech. “With the synthetic approach, you can start to think of the cell as a laboratory where you can tinker around and really ask questions about the basic principles of genetic circuit design.”

像该领域基础的工程师的意图一样，合成生物学也承诺帮助研究者以传统生物学无法实现的方式来理解细胞功能的基础，现任加州理工大学教授Elowitz说。“有了合成的方式，你可以把细胞当作实验室，并且思考和提出问题，了解关于基因电路设计的基本原则。”

The growing influence of engineering in biology is, in some sense, “the best of both worlds,” adds Church.The good design principles of engineering and the unique properties of evolving biological systems are “just an incredible combination,” he says.

生物工程的日渐增强的影响，从某种程度上说“ 对两个世界都有好处，”Church补充说。好的工程设计原则和进化生物体系的独特特征是“难以置信的结合，”他说道。