Regenesis (32 page)

Figure 8.1
Biobrick assembly

The assembly process, Knight commented in his paper, was such that “each reaction leaves the key structural elements of the component the same [which is what the “idempotent” of his title meant]. The output of any such transformation, therefore, is a component which can be used as the input to any subsequent manipulation. It need never be constructed again—it can be added to the permanent library of previously assembled components, and used as a compound structure in more complex assemblies.” This was standardization as implemented biologically.

Knight's idea of creating a “permanent library of previously assembled components” was the basis for the Registry of Standard Biological Parts, an inventory of snap-together, prefabricated genetic Lego blocks. Although Knight founded the library, much of its later development was substantially the work of Drew Endy.

When it was formally set up in 2003, Endy was a fellow in the Department of Biological Engineering at MIT. Like Knight, Endy did not start out in biology, and in fact had a love-hate relationship with the subject. He didn't much care for either the inherent complexity of natural organisms or their native tendencies to sporadically come forth with unexpected, “emergent” properties and behaviors.

“I hate emergent properties,” he has said. “I like simplicity. I don't want the plane I take tomorrow to have some emergent property while it's flying.”

What Endy liked about biology was that organisms were so versatile and pliable that you could coax them to build practically anything simply by manipulating their genetics.

“I like to build stuff,” he says, “and biology is the best technology we have for making stuff—trees, people, computing devices, food, chemicals, you name it.”

Unsurprisingly, Endy became a biological engineer, getting his PhD in biochemical engineering and biotechnology at Dartmouth. In 2003 Endy, who had won a teaching award at Lehigh in 1993, offered an independent activities period (IAP) course at MIT. Such courses were routinely given on offbeat subjects during the January break, and Endy's was definitely in the mold: Synthetic Biology Lab: Engineered Genetic Blinkers. Over the previous eight months, Endy had developed the course materials together with Tom Knight, Gerald (Gerry) Sussman of the AI Lab, and Randy Rettberg, who was a research affiliate of the AI Lab but had previously held executive positions at Sun Microsystems and Apple Computer.

The course, which was attended by sixteen students ranging from freshmen to doctoral candidates, was aimed at producing a genetic sequence that would make
E. coli
bacteria periodically emit a dim luminescence, flashing on and off like a stoplight (albeit one that flashed in exceptionally slow motion). Making bacteria fluoresce was nothing new in microbiology: genes for the green fluorescent protein (GFP) normally found in jellyfish were routinely spliced into an organism's genome for the purpose of creating a “reporter gene,” which signaled that a given molecular event had occurred within the organism. A blinking bacterium was nothing new, either. Michael Elowitz and Stan Leibler, then at Princeton, had created a three-part circular genetic loop in
E. coli
that made the bacterium oscillate regularly between “on” and “off” states. The gene for the on state also activated a green fluorescent protein gene that made the organism light up, a bacterium that blinked on and off. The controlling element of the object was a combination of oscillator and repressor genes, and so they called their new genetic circuit a “repressillator.” (Their report on the device was published in
Nature
in 2000: “A Synthetic Oscillatory Network of Transcriptional Regulators.”)

So, what was new about Endy's IAP-engineered genetic blinkers project in 2003? Basically, the idea was to turn the original genetic blinker into a new and improved version with superior functionalities. The oscillatory period of the Elowitz and Leibler blinker was measured in hours, which was longer than the cell division cycle of the bacterium itself. Consequently the state of the oscillator had to be transmitted down from generation to generation. So one subgroup of IAP students wanted to speed things up. A second subgroup wanted to create and install a “synchronator” gene that would make all the cells blink off and on in concert, like the lights on a movie marquee.

The students started with an initial biobrick parts kit put together by the instructors, Endy, Knight, Sussman, and Rettberg. The parts kit consisted of actual DNA sequences stored in freezers, sequences that encoded some of the most basic functions in molecular biology. There were promoters: sequences that started DNA transcription, and there were terminators: sequences that stopped transcription. There were antisense
RNAs
that blocked gene expression, and of course there were GFP genes that made cells fluoresce. A data book listed the function, structure, device name, description, and biobrick number pertaining to each separate genetic part. Essentially, the parts kit was an indexed bin of biological components and supplies.

Standard biological parts exist on three levels of complexity; the most basic is a genetic part such as a plasmid or a plasmid backbone. There are also composite parts, combinations of two or more individual parts, such as a reporter+quencher. The next level up is a genetic device, a group of components that perform a function of greater complexity, such as a device that initiates cell death or sends signals between cells. Finally, there are systems, entire organisms that do something—blink on and off, for example. (There are also “chassis” parts, which are commonly used lab strains of organisms such as
E. coli
, yeast, or
Bacillus subtilis
. These are more or less blank slate microbes that will receive the new genetic sequences and then express them as output.)

During the IAP course, the students came up with and contributed about fifty new genetic parts of their own, and entered each part's name, function, and other information in the data book. This was the basis for
MIT's current Registry of Standard Biological Parts, which can be accessed at
parts.mit.edu
. At the time the IAP course ended, there were about 140 annotated genetic parts in the registry. At the time of this writing there are over 7,000 parts in the registry, but this is changing quickly, since Harvard's team alone made 55,000 parts in the summer of 2011. Even the location and governance of the registry is changing, moving away from the MIT campus.

When the students were finished designing their new genetic circuitry, Endy sent the relevant sequence information to Blue Heron Biotechnology of Bothell, Washington, to be synthesized. The company, founded by MIT grad John Mulligan in 1999, found that half of the designs couldn't be produced, and those that could be synthesized didn't seem to work when injected into cells. And so the whole project seemed to be a washout.

But about a year later, Endy managed to get some of the designs to function as intended—just barely. “It would have been dumb luck if they all worked together out of the box,” he said.

The other instructors had their own postmortems. “It's interesting to think about where this is going, and of course we have no idea,” said Gerald Sussman. But Tom Knight did have one: “Hopefully, the next IAP students will get
E. coli
to blink ‘dash-dash, dot-dot, dash' (Morse code for ‘MIT').”

The course was repeated in January 2004, with a project named Engineered Genetic Polka Dots. Student teams created new standard biological parts and designed genetic circuits that made cellular patterns including polka dots (the team in question called itself the Polkadorks) and bull's-eyes, as well as an ambitious plan to make cells swim together like a school of fish.

In the end, none of these second-generation grand designs behaved as planned, either. Evidently, putting biobricks together and getting them to work successfully would be slightly more challenging than anyone had thought.

But then came iGEM.

In the beginning, iGEM stood for intercollegiate genetically engineered machines. The idea was for MIT to bring other American colleges into the
business of engineering genetic parts and devices, and to stage a competition among the participants. The competitions would be modeled on student robot contests, events that were so successful at drawing in students that the prize ceremonies at some of them filled entire stadiums. So Rettberg, Endy, and Knight rounded up Caltech, Boston University, Princeton, and the University of Texas-Austin (where Endy had spent some time as a postdoc) and delivered them all a one-phrase challenge: design and build a genetically encoded, finite-state machine (one that transitions from one state to another under the control of a program).

The students would start work in the summer of 2004, continue through the fall, and then meet up for a jamboree in Cambridge early in November to compare and share their results. Which is indeed what happened.

The Princeton team set out to build the biological equivalent of a children's game called Simon, a test of memory in which participants had to repeat a pattern of signals that grew in length and complexity with each successive iteration. So the students designed a three-cell system that, when it detected a particular sequence of inputs, would respond by triggering a release of the reporting molecule, which in this case was yellow fluorescent protein (YFP).

The Caltech team wanted to design and build a strain of yeast that could detect three different levels of caffeine in coffee. Boston University teamed up with Harvard to design cells that could be made to count. The UT Austin contingent set out to build what sounded like the neatest bacterial app of them all, an
E. coli
film that would take what amounted to a biological photograph.

As it turned out at the jamboree, the Princeton team couldn't get its YFP reporting mechanism to work. The Caltech students, by contrast, brought off their project handily. Their engineered yeast glowed green in the presence of low caffeine, green and yellow in medium caffeine, and yellow alone in high caffeine. In a campus coffee shop, the yeast successfully identified decaf, regular, and espresso coffees. The Boston University and Harvard team eventually got their bacterial counter to work and published a report on the project (together with considerable subsequent work) in
Science
five years later.

The stars of the show were the Texans, who added photoreceptor genes to
E. coli
, created a thin film of the light-sensitive bacteria, and got it to display the phrase “Hello World” in the form of deposited pigments (
Figure 8.2
). This phrase has been a standard simple test for computer programmers and an inside joke ever since Brian Kernighan wrote a Bell Labs memo in 1974. The experimental result was published in
Nature
: “Synthetic Biology: Engineering
Escherichia coli
to See Light” (2005). A behind-the-scenes story is that the original pigment patterns were due to the action of the light directly on the colored nutrients in the agar. In other words, the photo was a product of the nutrients rather than the bacteria. The team had not done the control step of leaving out the genetically engineered microbes. Nevertheless, that photo was all over posters representing iGEM for the next year! But this lapse was repaired before the paper was submitted to
Nature
. (Team members also sharpened the fonts and added the word “nature” as well as faces of many almost-famous people, like Andy Ellington of Austin, Texas.)