I am one of the creators of the first "synthetic" bacterial cell. AMA

Can you give a summary of how the genome's created?

Is it assembled "from scratch" or is most of it borrowed from a pre-existing cell or cells.

If it's made from scratch, how is this done?

All good questions deserving of upvotes.

Briefly, the genome was assembled in four stages: 1kb, 10kb, 100kb, and complete. The entire M. Mycoides genome is about 1.1 Mb long - this means 1,100,000 base pairs of DNA. It was first divided into chunks of roughly 1,000 bases. These 1,078 "1kb" pieces were ordered from a DNA synthesis company. 10, 1kb pieces were assembled using an in-vitro technology to produce the 110, 10kb cassettes. 10, 10kb cassettes were then put together using a technique called yeast homologous recombination to yield the 100kb segments. The final assembly step (of combining 11, 100kb segments) is also done in yeast, taking advantage of their natural ability to stitch together overlapping pieces of DNA.

To answer your 2nd question, the genome is assembled "from scratch" in the sense that the DNA itself is synthesized using a machine, and then put together in a stepwise fashion. The sequence of the DNA, however, is almost identical to the native M. mycoides genome. So the genetic information is indeed borrowed from an already existing species, and the genome transplantation process also requires a recipient cell that is very much pre-existing.

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I'm glad you asked this question.

The short answer is not much is actually needs to be done to prepare the recipient cell. The cell and the synthetic genome are incubated in together in a solution which encourages cells to fuse together. One of the key ingredients is "PEG" (polyethylene glycol) - a polymer which acts as a crowding agent, and essentially pushes cells into clumps. Like two oil droplets coming together in water, the plasma membranes of M. capricolum cells can be rearranged and merge with one another. During this shuffling process, the synthetic genome occasionally finds its way into a cell. Note that this is a rare occurrence, and only about 1 in every 150,000 recipient cells will be transformed. The biophysics behind this are not well understood, and it's an active area of research at the JCVI.

Once inside a cell, the synthetic genome starts to express its own genes. You are correct in your hunch that antibiotic resistance (to tetracycline) is used to select for transformants. There is no need to destroy the host genome, it is simply lost after a round or so of cell division. Any cells which do not contain the synthetic genome will not be expressing antibiotic resistance genes, and won't survive.

I understand using tetracycline resistance to select for transformed cells, but won't cells that have this new genome still be transcribing their original genome? How is the original genome lost after a few rounds of cell division, and how do cells divide with two separate genomes?

Again, the process is not known in complete detail. Genomes take up a lot of space in the cell, and are metabolically expensive to replicate and maintain. So one theory is that cells containing only the synthetic genome quickly out-compete cells containing both.

Also remember that the cell cycle naturally involves copying all of the DNA in a cell. Two separate genomes briefly exist in a single bacterium, before division occurs and the genomes are segregated into two daughter cells.

I was watching a TED talk by Venter where he mentioned something about the donor and recipient genomes having different restriction systems making it possible to have the donor strain destroy the recipient DNA. I see from your post that you didn't use this system, but would you think that it would improve the efficiency?

Also, I'd like to ask if you are now planning to build a completely designed genome? And with that I meant that it wouldn't be a replica of a known genome but that it would only have selected genes.

• I'd like to ask if you are now planning to build a completely designed genome? And with that I meant that it wouldn't be a replica of a known genome but that it would only have selected genes.

In the long run, yes, it will be theoretically possible to generate genomes which are unlike anything that has ever existed in nature before. One limitation to this is that we still need to have a recipient cell which is similar enough that it will tolerate the synthetic genome. We can test this boundary in the short term by adding and deleting genes in small steps.

Is this similar to the phenomena where some bacteria will absorb DNA that is just floating around in the goo? All I can think of is that freshman bio lab where we made bacteria bioluminescent by having it absorb jellyfish DNA.

It is similar in principle, but at a much larger scale. Many bacterial species are equipped with mechanisms for taking up DNA from the environment - this is one way in which antibiotic resistance spreads. Taking up an entire genome, however, does not occur in nature.

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Yes, we do hear a lot of that.

One advantage to building your own genome is that there is no limit on the number of gene additions or deletions you could introduce into the synthetic design. A single gene knockout might be a trivial task (if you're working in a microbe with established genetic tools), but when you want to delete hundreds of genes at once or introduce entirely new metabolic pathways - building from the ground up starts to make sense.

We have now paved the way for a new scientific discipline - which might be called something like "combinatorial genomics." We envision creating libraries of synthetic genomes - each containing differing numbers, arrangements, and types of genes. These genomes can be pitted against each other in a high throughput manner to rapidly identify which combination produces the best results for, say, making ethanol.

So did you write that 1.1 Mb from scratch. I understand most of it is identical to existing bacterias, so my question is rather: does every single part of code make sense to you? Because all living organisms are a product of evolution, their genetic code would be a hack upon hack. If you try to intelligently design it instead, there's a possibility you could make it several times more efficient? On the other hand bacteria had plenty of time and opportunity for optimisation. So is there still room for optimisation in those 1.1 Mb?

The entire 1.1 Mb genome was synthesized from scratch. However, the genome that we created is almost an exact duplicate of the naturally occurring M. mycoides genome. This means, for example, that (except for the regions we added to or deleted from the genome) all of the genes occur in exactly the same order as they do in nature.

The majority of genes in M. mycoides do "make sense" to us, but there are still a few of unknown function. The genome is already quite small and very efficient. We could intelligently design an even smaller genome, but the trade-off would be a decrease in survivability. M. mycoides is indeed already optimized to do what it does best - living in goats. :)

Complete noob here. I was wondering... What is the point at which the DNA starts 'functioning'. Does that happen at some stage during its assembly? Or when it is put inside a recipient cell? Can you actually see it starting to 'work'? Thanks for the AMA btw.

edit: typo

Good question, especially for a complete noob.

During the assembly process, fragments of the synthetic genome are "stored" in both E. coli and in yeast. Yeast and bacteria use different machinery for expressing their genes, so the genome is not functional at these assembly steps. It's possible that some gene expression occurs in E. coli, but Mycoplasmas use a different codon system and the products would probably not be functional.

The synthetic genome only really begins functioning when put inside the environment of the recipient cell - which is a close enough relative for the genes to be turned on. One of the first signs that the genome is actually working is the blue color change, like you see here.

I'm not at all familiar with this, but let me try to rephrase that to see if I understand.

First if all, you came up with a 1,100,000 series of Gs, As, Ts and Cs, each one representing a base pair. The sequence you chose was actually an almost exact copy of the existing DNA of another species, but you did a little bit of editing on the string to tweak it / watermark it.

You then broke this 1,100,000 letter long string into 110 substrings, each one about 10,000 pairs long. Then you broke these 10,000 pairs into strings of 1078 pairs. These 1078 pair strings were the basic building blocks of the DNA strand you assembled.

A DNA synthesis company out there can provide these basic building blocks, so you ordered all the chains you needed from them.

Once you had these building blocks, you used in-vitro technology to string them together into 10,000 pair long "cassettes". Each of these "cassettes" was made from 10 building blocks.

The 10,000 medium-sized blocks were combined, 10 at a time, to form 100,000 pair long strings using "yeast homologous recombination"

These 100,000 pair long strings were slightly longer than they needed to be so that they had "header" and "footer" sections. You used yeast to combined these 100,000 pair long strings together, taking advantage of the fact that yeast would take the "footer" of one string and attach it to the "header" of another string if the header and footer matched.

Is that roughly right? If certain parts are wrong, what did I miss?

What's the yield on each step? For example, say you coded the 1kb strings by letter and wanted to produce the sequence ZXIZXIPGDA, where each letter represents one of the 1kb pieces. What fraction of the chains that you started with actually end up in a ZXIZXIPGDA chain? Most of them? Very few? Do you basically toss them together and hope for the best, filtering out all the ones that fail, or do you mostly get proper chains, with a few failures that have to be picked out?

Similarly with the other steps, how often did you get sequences that weren't what you were after vs. ones that were what you wanted?

You've got the basic idea down pretty well.

One clarification about the header and the footer idea, just so there's no confusion. If your cassette 1 was "ABCDEFG" then your cassette 2 would look something like "FGHIJKL." When you stitch these two together, you get "ABCDEFGHIJKL." and not "ABCDEFGFGHIJKL." This is just a consequence of the way that the homologous recombination works. Also, note that the headers and the footers are all parts of the genome itself, and not some extraneous pieces of DNA used only for stitching things together.

When we were assembling the 10kb cassettes, we would screen each one to ensure that it was the correct size. Sometimes the yield was 100%, sometimes we would have to screen dozens of colonies before finding one with the correct assembly.

When you finally did it, was there a crack of lightning and someone shouting "ITS ALIVE ITTSSSSSS ALIIIVEEEEEEE" ?

hmmm?

ಠ_ಠ

How, exactly, does one watermark a genome with "a coded version of the names of the team members, a URL, and an e-mail address"? How do you know those segments of the genome are inert? How many generations will they last before mutating? And is this all a devious plot to make the scum in my shower spell out your logo you evil mad scientist you?

The genome contains only As, Ts, Gs, and Cs. In living organisms, three bases in a row are called a "codon" and specify an amino acid. The 64 possible codons encode stop/start instructions and only 20 amino acids. In other words, there is a lot of redundancy. So, I think you could imagine a system where each codon stands for a different letter of the alphabet, plus plenty of extra codons to cover symbols and punctuation. That is how the watermarks were designed. We know these regions are "inert" because they do not contain promoter sequences or ribosomal binding sites and thus won't be transcribed/translated.

I got all excited about the watermark - as i think joyce is a cool guy isn't afraid of anything etc. anyway. I got the supplementary info, got the watermark sequences, translated into aminoacids into all the frames and still can't find the quote - am i missing something? is it coded more than just amino acid letters?

also, is your name in the watermark?

Perhaps we didn't use the traditional genetic code? That would be too easy to decipher. :)

In order for me to understand how much of a breakthrough this is let me pose my question in the form of a thought experiment:

Imagine we had a sophisticated group of scientists leave earth and colonize another planet somewhere lightyears away.

Now after they leave imagine that a new bacterial cell is found on earth that has massive benefits for humanity.

Would it be possible to send to the colonists all the information necessary to reconstruct this beneficial bacteria from scratch?

In other words, could you effectively turn the entire bacteria into pure information, broadcast it across space and reconstruct the bacteria at some remote destination?

The sequence of the synthetic genome is indeed stored purely as information on computers. Nothing but ones and zeroes. If you sent this sequence to a DNA synthesis factory on the moon, they could make it for you using the same techniques we developed. The problem is, you still need a recipient cell to put that genome in to.

Think of it this way: the genome is like a blueprint for "how to build M. mycoides". It contains all the instructions necessary, but you still need to hire some engineers and construction workers who know how to read that blueprint and execute it. The recipient cell provides a cell membrane to encapsulate the genome, along with the protein synthesis and DNA replication machinery needed to "boot it up."

Of course, if your scientists are smart enough to colonize other planets, they may have already come up with ways to artificially synthesize all of the necessary cellular components as well.

Does the synthetic genome include the histone code, or is this purely the nitrogenous base code?

I was under the impression that some relevant information is encoded in the from of histones (methylation tends to repress the transcription of that region) and that purely examining the bases will give an incomplete picture.

Good question, but remember that bacteria do not contain any histones. Histones are only used to organize DNA in eukaryotes - which have much larger genomes.

How long will it take us to develop a synthetic cell that will perform useful tasks? Clean up oil in the gulf or clean my arteries?

Good question. Everything awesome in science always seems to be "just 5 to 10 years away!" - which leads to disillusionment from the public when overly optimistic projections inevitably fail. Thus, I'm not going to speculate about exactly how long it will be before useful applications emerge. In general, the most promising research in Synthetic Biology is in the bioenergy arena.

Here are some relevant news articles, in case you have been living under a rock today.

JCVI Press Release

BBC

NYT

Do you believe in god?

That's an easy one, nope!

What an amazing achievement.

What's something that is even more amazing than people think?

How about something that you didn't know if it would work, but it did?

What's a limitation of the technology that isn't immediately apparent?

• What's something that is even more amazing than people think?

Forgetting about the transplantation, the synthetic genome itself is the largest man-made molecule of a defined structure ever created.

• How about something that you didn't know if it would work, but it did?

In the beginning, there was never any guarantee that they synthetic genome would ever be able to be "booted up" in a recipient cell. The transplantation step is truly unprecedented in the history of molecular biology.

• What's a limitation of the technology that isn't immediately apparent?

The "recipient cell" (M. capricolum) is a very closely related species to the "donor cell" synthetic genome from M. mycoides. It's not yet clear just how different the donor genome can be for the transplantation step to still work.

What do you think the odds are that you can insert a gene of choice and have it work as expected (e.g. a florescent gene)?

Do you think it will take a lot of trial & error?

Will it beat the shotgun approach to gene insertion in terms of costs?

If you have a gene of known function that has successfully been expressed in bacteria before (such as GFP), the chances of it working as expected are virtually 100%. For example, in the synthetic genome a gene called "lacZ" was included. This gene (well known by molecular biologists) encodes the enzyme beta-galactosidase, which is used in the metabolism of sugars. It also converts a chemical called X-gal to a blue compound, which is why the pictures you saw on the BBC looked like blue eyes. Native M. mycoides or M. capricolum cells do not make any beta-galactosidase, so if the colony is blue you know it contains the synthetic genome. (probably)

Can you elaborate on the way the DNA was made?

I can tell you we outsourced the assembly of the 1kb cassettes to a company called Blue Heron Technologies. This step alone had a price tag upwards of $1 million. I am unaware of the technology actually used to synthesize the DNA, and I suspect much of it is proprietary.

I suspect much of it is proprietary.

Well that sucks. What are these cassettes? Like, are you popping a tape in the bacteria and then it plays whatever was on it?

A "cassette" is just a colorful terminology we use when talking about a specific piece of DNA. All of the segments of DNA were numbered in order to make keeping track of them all feasible. So during the first stage of assembly, you might refer to "cassette 1-10" , in the second stage "cassette 1-100" , and so on.

Also, it's not a crime against humanity that the DNA synthesis technology is proprietary. The machines that preform these reactions are very expensive - so it's commonplace in molecular biology labs for DNA synthesis services to be done by an outside company.

How much time does your lab spend discussing possible ethical ramifications?

Also, how much time does you guys spend discussing really out there hypothetical nightmare situations?

This is a really important question, and I want to assure you that the JCVI has been concerned about the societal and ethical implications of its work from the very beginning of this project 15 years ago.

The research has undergone ethical review and risk analysis from independent scientific and governmental organizations such as the University of Pennsylvania, MIT, the National Academy of Sciences, the Department of Energy, the Department of Defense, and National Science Advisory Board for Biosecurity. More information is available here, see the "Fact Sheet: Ethical and Societal Implications/Policy Discussions about Synthetic Genomics Research."

These reports share a common theme: a marginal increase in risk (as compared to already well-established research in the field) and an exponential increase in the potential benefits to society.

I'm not sure if this has been asked before, and I'm not sure if this is a stupid question, but I'm curious, so here goes.

What happens if you generate a random sequence of DNA pairs, assemble it synthetically, and transplant that into a host cell? Would you create a new species? I have the vision that in several years we will have Spore game In Real Life.

No such thing as stupid questions. :)

If you generated a random sequence of DNA, it would be very unlikely that the product would code for anything useful - and many times more unlikely that it would be a viable new species. As an analogy, think about throwing together a random sequence of letters, and how improbable it would be that you would ever create a novel.

Your "Spore" vision, however, is not so far fetched. Now that we have a synthetic genome, we can drop in new genes at will to change the cell's properties.

do you know craig venter? if so, what's he like? he always struck me as a pretty cocky guy who's in it for the money and to have his name plastered everywhere.

Honestly, I have only met Craig a handful of times. I worked in the Rockville branch of the JCVI and he was very rarely there. (Someone of his caliber is not involved the day to day operations going on.) When I did interact with him it was always a pleasant experience. At least when it comes to interacting with his employees, he didn't appear to be a bad guy.

I don't know if this question makes sense but here it goes:

How difficult would it be to create a living cell with just the bare minimum of DNA for it to be considered "living". The "most basic cell possible" in other words?

Great question.

The JCVI has been working on finding the so-called "minimal bacterial genome" for over a decade. The first synthetic genome we created came from the species Mycoplasma genitalium, and was chosen because (at 580kb) it is already among the smallest known bacterial genomes. From there, we preformed a series of knockout experiments and found out that, amazingly, only 382 out of the 482 genes found in M genitalium were actually essential. As a point of reference, humans are thought to have somewhere in the neighborhood of 20,000 - 30,000 genes.

With synthetic genomes, it will be relatively easily to iteratively drop out more and more genes and test what the minimal set of genes needed to sustain life will be. One caveat, of course, is that environment matters. The minimal cell would be grown in conditions that provide all the nutrients it needs. Thus, certain metabolic genes become "non-essential," but the minimalist bacterium wouldn't be able to survive on its own.

How do I get to do research like this some day? I would LOVE to do something like this for a living! ps. Great Job!

I'm not sure what level you're at right now, but here's a short answer: stay in school. Get good grades and apply for the best colleges that you can get in to. Don't be afraid to try new things, or even to do seemingly drastic things like changing your major midway through. The most important thing is to find something that you are passionate about. If you do end up in a scientific field, start doing research as soon as possible in your undergraduate career to pave the way for graduate school.

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Interns at the JCVI put in no more than 40-50 hours a week. We want to get people interested in and passionate about their projects, not kill them. Even graduate students aren't expected to work 70+ hrs/week, yeesh!

Do you have any comments on the press stories about your research? Have there been any frustrating misrepresentations?

I think it's frustrating that every news article I've read has the basic format of "X happened, but scientist Y considers it not that important, and group Z finds this highly unethical and dangerous."

X, Y, and Z are all valuable things to talk about, but they don't deserve equal time. With the very limited amount of space available to convey scientific advances to the public, I wish more energy was put into attempting to explain exactly what we did.

So how long until you synthesize me a pair of laser eyes?

Sorry, I can neither confirm nor deny the existence of a laser eye project.

Do the synthesized cells reproduce?

Let me clarify that the "synthetic" part is only the DNA that we have built completely from scratch. (Even this is arguably not artificial, since it was copied from a living species.) The synthetic genome was then transplanted into a wild-type recipient cell, which was a closely related species found in nature. In order for the genome to work, it needs the machinery already existing in the recipient cell to express itself. It's an imperfect analogy, but you could think of the synthetic genome as "software." The software can't do much on its own without the "hardware" which is capable of reading it.

That being said, yes, once the synthetic genome is "booted up" the cells do in fact reproduce.

I haven't read the paper yet, but did you use a MAGE like platform to insert in DNA at bit by bit into a BAC?

Propagation of fragments of the synthetic genome occurred in E. coli using BACs as long as possible (below 100kb), and then moved into YACs.

Do you think it would be worth buying stock in Craig Venter's company?

Sorry, Synthetic Genomics is not a publicly traded company.