Going with the Flow in Replicons

RNA replicons are an exciting new platform for synthetic biology.  Basically, you take a virus and you remove the part that makes it infectious, replacing it with whatever synthetic system you want to put into the cells instead.  You leave the part that replicates inside the cell, though, so that even if only one or two pieces of RNA get into the cell, they’ll self-amplify and express your system really strongly.  It’s great because you can get expression as strong or stronger than you’d get from editing it into the cell’s DNA, but it’s safer to use because it doesn’t get into the DNA.

We’ve already shown that you can precisely engineer unregulated expression from replicons.  Regulating expression, to actually control which parts of your system are running when, is trickier.  Even though people have been able to get repressors like L7Ae working on replicons, they are much less effective than with DNA, even though they’re being expressed more strongly.  This is a problem if you want to make controlled replicon systems, and seems like it really ought to be solvable.

Digging into this problem, I found that indeed, it looks like the problem is that when you set up a system to work on DNA, it’s likely to work poorly because it’s going to end up fighting against the natural dynamics of the replicon.  Turn that around and go with the flow, however, and it looks like it should be possible to get even better performance on a replicon than you can on DNA.

In my talk at IWBDA this year, I showed my conclusions about why things go wrong when you just go straight from DNA to replicon: since everything is amplifying exponentially as the RNA replicates, low levels of expression get raised a lot higher, making the system “leaky,” and outputs rise before there’s enough regulator to shut them down.  Big problem.

Model for L7Ae repression of mVenus from replicon, providing an explanation for observed poor performance.

Once you’ve identified the problem, though, some good paths to try towards fixing it appear as well.  In this case, it turns out that decreasing the dose of repressor and increasing the rate at which the output decays looks like it should radically increase the efficacy of repression.  Will it really do so?  We won’t know until it can get tried out in the lab, but the model’s based on things that have worked before and there’s a nice broad area of high performance to shoot for, so I’m awfully hopeful this will work.

According to the replicon repression model, the best performance comes when both L7Ae dose and mVenus degradation time are moderately reduced.

Biological Design in the English North

This past week was the 8th International Workshop on Bio-Design Automation (IWBDA), along with a couple of other associated events—a tutorial day on the Synthetic Biology Open Language (SBOL) beforehand and a workshop on the integration of electronic and biological design automation afterward.. All told, I spent nine days in England (and Scotland!), immersed in synthetic biology for almost the entirety of that time, and though it was all good, it was also quite exhausting.  I’ll come back to the technical content on another day.  For now, I want to talk about Newcastle, where IWBDA another these other events were held this year.

This is my third time in Newcastle, and if I wasn’t cutting down on my travel, I’d be back there again in a month for another meeting on synthetic biology standards (I’ll be dialing in instead). There’s an excellent group of synthetic biologists there, a big node in the larger UK network, and I work frequently with Anil Wipat on SBOL, he being both a long-time contributor and the current chair of that standards effort.  I always enjoy spending time with him and the rest of the Newcastle crew, who, like me, are true believers in the power of characterization and design tools in wrestling with biology.

As a city, I find Newcastle upon Tyne to be a delightful hodge-podge of the old and new.  From its ancient military history, it eventually became a tough old industrial town and port city, one of the anchors of the English North.  Rail lines tangle together at its center, at the top of a steep embankment beside the river, and spider out across bridges in all directions.  One route goes over a remarkably high rail bridge, standing far above more modern road bridges and a beautiful walking swing bridge just a bit further downstream.  An tall old mill has been repurposed into a modern art museum, and on the high side of town the university stretches between two broad lanes of park.

High Level Bridge

Gateshead Millenium Bridge

The Long Stairs

The people I see on the streets of Newcastle are a motley hardy lot, young women walking apparently imperturbable through the freezing rain in their miniskirted clubbing outfits, tough geezers yelling to each other across the streets at four in the morning, loud music in every pub and restaurant.  Joggers rush by the cows in the parkland past the university, and photographers set up on the riverbank talking amiably to one another by the unhappiest looking palm trees I’ve ever seen, their bark and leaves looking shriveled and grey in the cold. I definitely am an outsider there, but so do many of the others seem to be, and I’ve never yet been hassled for it.  It’s feels to me a much less urbane and international sort of crowd than one sees in London, but a rough diversity that I find I do appreciate.

All in all, a good place to wander through with one’s head drifting around and processing all the science of the day.

Traveler's Blues

Today, I stepped onto my 60th airplane of the year.  I'm on my way to celebrate my cousin's wedding, which is fun and awesome, but all the same, I'd prefer not to have such a comfortable familiarity with airports right now.  The Cedar Rapids gate personnel and I are starting to recognize one another and learn one another's names.

Wheels down for landing at O'Hare

Sixty airplanes in 24 weeks: that's more than two airplanes per week.  Of course, it's a bit more bursty than that, since flying out of Iowa means I basically always connect, so each trip is generally four flights.  Still, that means on average I'm taking a trip more than twice a month, and I'd really prefer to not be doing that.

It's hard to say what I would drop in order to reform my life to be more stationary, however.  Individually, every one of those trips has been a good idea.  Collectively, I am saturated.  When I am at home on a weekend, I don't much want to go anywhere or do anything.  Hang out with my wife and daughter, go for a long walk in the cornfields or the woods, read silly articles on the internet.

It feels almost shameful to me to even voice this complaint, since at its root is having a career that seems to me to be going quite well, that affords me opportunities to go all sorts of places and talk to really awesome people about things that I find interesting, and that also provides me with resources enough to go to visit my relatives and friends on special occasions.  These choices in my life, however, do come with costs, and those costs exact a toll on my body, mind, and spirit.  

I'm happy that I'm flying to Maine right now, and I also wish that I could be snugged up at home on the couch, scritching a cat curled up in my lap and contemplating nothing in particular.

Podcast on adoption of SBOL by ACS Synthetic Biology

Following up on my recent post announcing the official adoption of SBOL by the ACS Synthetic Biology, the journal has just posted an interview with me and Nathan Hillson on SBOL.  Have a listen and enjoy!

A big step forward for engineering in synthetic biology

Today the synthetic biology community took a big step forward in its progress towards being a mature engineering discipline.  As of today, ACS Synthetic Biology became the first scientific journal to officially adopt standards for how genetic constructs should be depicted and how the designs of engineered organisms should be recorded and shared.

ACS Synthetic Biology chose to adopt the two current Synthetic Biology Open Language (SBOL) standards.  The journal's author guidelines are being adjusted to state that genetic constructs should be depicted using SBOL Visual and that SBOL 2 is the preferred format for nucleic acid sequences.

• SBOL Visual, which I've written about a couple of times, gives a palette of symbols for making clear and precise diagrams of genetic regulatory networks, just like electronic symbols let us communicate clearly and precisely about circuits. 
• SBOL 2, which I've also written about before, is a data model for communicating the full blueprints for a genetic design, both structure and function.

Wider adoption of standards like these is critical for moving the engineering of living organisms from being a delicate craft, practiced with great difficult and uncertainty, toward becoming a familiar and reliable part of our capabilities as a species.  Effective engineering demands clear communication and the ability to integrate components from many sources.  Safe and ethical engineering practices demand them even more so.

As a species, we've made the transition from guild craft to engineering discipline with stone and iron, with bridges and buildings, with electricity and even radiation. Step by step we are on our way to doing the same with biological organisms.

Why I rejected your scientific paper

A little while back I stumbled across a post by Matt Welsh entitled "Why I gave your paper a Strong Reject," and it's had me thinking since then about my own pet peeves as a reviewer.  One in particular has been frustrating me most of all of late, however, and since it strikes at the heart of one of the most difficult yet simple aspects of science, I thought that I might write about it here.

Ultimately, all of the scientific method boils down to one simple question: "How do you know?"  This is the core of every scientific paper, and when I do not find it, I am guaranteed to recommend rejection rather strongly.  I've had a whole string of papers to review that fail in this regard recently, and it frustrates me to the point where I have changed the way in which I read review papers.  When I begin to review a paper these days, I start by looking at the title and abstract, then immediately flip to the end of the paper to see if there are any results.  Whether it be real-world evidence, lab experiments, simulations, or theorems, I want to know that the authors have delivered something that gives substance to the discussions and assertions that are presented in the rest of the paper.  If so, then I will read the paper with much more enthusiasm and give it a careful review; if not, then I will skim quickly to make sure that I have not missed anything fundamental, but with the pre-judgement that the paper is almost certainly going to fail and be rejected.  Even a review paper would not completely escape this judgement of mine: after all, there should be some synthesis that brings all of the different pieces together to differentiate a good review paper from merely being a keyword search.

So far, it's simple.  You can't claim to know something if you don't present some sort of information to justify that knowledge.  Where it gets difficult is that justification of knowledge is often quite a different matter than people seem to think it is---even scientists.  For example, people often present a number in isolation as though it is meaningful, without giving context to compare it to.  If I tell you a current car can drive 60 miles/gallon, that's a meaningful number because you probably know what other cars tend to get; if I told you that a certain type of car in the 1930s got 12 miles/gallon, however, would that be a lot or a little comparatively?  I certainly don't know, and I get frustrated when people drop context-free numbers like this.

This applies not just to obscure bits of scientific inquiry, but also to all our ordinary lives.  News articles, for example, are full of assertions based on numbers without comparison, as are the labels on foods we eat.  Is $10 million dollars for a bridge a crazy boondoggle or remarkably efficient?  Does "low fat" just mean higher sugar and salt, or that it's actually made in a more healthy way?

At the same time, this doesn't mean one should fetishize any particular classical mode of experimental design or demand "absolute proof" of anything.  There's a lot of ways to lay one's hands on information, and most of them do not need to be double-blind controlled randomized trials.  My own scientific work, for example, almost never involves blind trials, because I work with strong signals and can use computer programs to ensure regularity in how each piece of data is treated.

But dammit, at least give me something to support your castles in the air!

DNA hard drives?

A big change may be coming to computing, and a very strange one at that: sometime in the not too distant future, we may find ourselves storing most of our data in biological DNA rather than our conventional silicon and magnetic storage.  I'm in the midst of a two-day workshop that's investigating this possibility, and there's both an interesting prospect and some very big challenges to overcome.

The core idea and concern is that we're just about at the end of what we can do with silicon. For the past fifty years, the density of transistors in silicon-based computing hardware has doubled approximately once every two years, an observation known as Moore's law.  This observation gave rise to a self-reinforcing market structure and the rapid expansion of cheaply available computing has transformed every aspect of our civilization, binding us ever tighter into a connected global society for better or for worse.  That ongoing increase will soon reach the physical limits of what can be achieved with silicon, however, and one of the interesting directions that people are looking to do better is organic materials like DNA.  The theoretical storage capacity of DNA is about a million times denser than flash drive memory, simply because the molecules involved can pack information that much more densely.  To give a sense of scale, that means that you could (in theory) pack one of the gigantic warehouse-complex data-centers that places like Facebook and Amazon build into a single smallish room.

The goal of this workshop is to figure out just how obstacle-strewn is the path between that theory and reality, and figure out whether it's worth pursuing.  This is another one of those interesting life-on-the-interface meetings, with biologists, chemists, computer scientists, information theorists, and silicon specialists all colliding their different viewpoints; with academic theory, government program-building, and corporate pragmatism all sharing a space.

If these DNA hard drives happen, they'll probably be like tape archives, rather than fast computer drives, but they'll be able to store so much information that would still be very useful to consider. There's a fundamental market challenge in that it's not clear whether DNA synthesis technology has enough pull to support the "writing" part of the hard drives.  There's also a long gap between the density of DNA and the realities of making a reliable storage appliance (error correction, packaging, input/output channels, keeping out biological organisms that would like to eat it, etc.), which I estimate will probably cost about two orders of magnitude, giving only about 10,000-fold improvement over the limits of silicon---which is still quite a big deal.

This is not the main direction that I am going with my own research, which is much more about controlling living cells, but it's quite interesting to be asked to be involved in, and close enough that there may be use for the things we've been developing elsewhere.  Plus it's just plain old fun science fiction and I'll be very interested to see just how it can be developed.

Congratulations to Swati Banerjee Carr!

On Monday at Boston University, I attended the successful Ph.D. thesis defense of Swati Banerjee Carr, who I have been co-advising with Doug Densmore for the past couple of years.

Swati's work is, to my thinking, a classic case of how a lot of science doesn't start with "Eureka!" but with "That's funny..." or, as in Swati's thesis: "Why doesn't any of this work?!?!"  Originally, she was building a simple test system to characterize various different pairs of repressors and promoters in E. coli.  These are some of the most basic and common building blocks in synthetic biology: a repressor acts on a promoter to suppress the gene controlled by that promoter, which means that controlling repressors can switch things on and off in a cell, including other repressors, making it one of the foundational tools for controlling the behavior of cells.  Practically everybody uses them, and in her system she was using some of the most well-known and best understood biological parts around, and yet when she put them together nothing worked.  When she put the parts together in different orders, however, sometimes it worked and sometimes it didn't.  "That's funny..."

With a bit of study, including results of some of my other recent work, the key problem was identified as being the bits of DNA just before the promoters, which changes depending on what else is in the system and how the parts are arranged. And so, Swati's project ended up shifting away from the original plan and focusing instead on solving that problem of context dependence. Now, at the end of her doctoral work, our work together has resulted in a lovely protocol for creating "upstream insulators" that make a promoter behave pretty much the same no matter where you put it.  The data is really beautiful, but I can't share it quite yet: not until Swati has officially deposited the thesis.  What it shows, however, is a remarkably stark contrast:

• Without insulators: a genetic circuit in which every permutation is different, pretty much none of them are "working" by even the most generous definition, and there's not even any real pattern to the chaos
• With insulators: every permutation does almost exactly the same thing, with quite strong and consistent signals for all of them.  

I think this has the potential to be quite a big deal, and don't know why anybody would ever use an uninsulated promoter again once this gets released.

Swati gave a good talk to a packed room, standing room only.  She's got some homework from me and the others on the committee, to improve her actual thesis document some more before it can be considered quite complete, but she's on the home stretch, having done some damned good work, of which I think she can rightly be quite proud.

Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli

I've talked about the interlaboratory studies we've been running through iGEM previously, and while I've been quite excited before, now is the biggest news I have to announce yet: our paper on the results of the studies has just been published in PLOS ONE: Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli.

Fluorescence from iGEM interlab constructs (Credit: Oxford iGEM 2015)

In sum: over the past two years, teams from nearly 100 institutions around the world measured the same simple genetic constructs for expressing green fluorescent protein, and this paper reports their results, crediting all of the more than 600 iGEM authors involved, including lots of undergraduates and even high-school students. In my eyes, the two key results from this study are:

• Ratios between strong fluorescence were remarkably precise.
• Weaker measurements were extremely unreliable, but the problem does not appear to be the biology!  Instead, it appears to be differences in how people use their instruments and handle their data.

These are really good news, because it means that some of the well-known problems in understanding and reproducing biological research might be tackled simply by improving our ability to calibrate our instruments and communicate about our measurements.  That's hard, but it's a lot better than thinking that biology might just be inherently too messy to understand properly.

We've also published all of the raw data submitted by all of the teams, so that people can dig further into the data if they're interested and see what else may be lurking there.

And what will 2016 bring?  That is still in planning, dear reader: you'll just have to wait and see...

How studying synthetic biology has improved my health

A curious thing has happened to me recently, which I would like to share with you, dear readers: over the past year, my study of synthetic biology has significantly improved my personal health. Now, don't worry, I'm not eating poorly tested experimental organisms to improve my gut microbiome or anything like that.  In actuality, the connection is much more indirect: my engagement with biology has simply led me to rediscover something that had fallen from awareness in the course of my modern life.

The story actually begins with the negative impact of science on my life, more specifically the effects of stress and frequent travel.  I am a person who has been overweight for much of my life, and not just by the terrible body-mass-index (BMI) measure that is typically used to judge such things.  At my height, BMI says that I should be carrying at most about 175 pounds, which is simply something that I do not believe.  My ancestry has gifted me with broad shoulders and a solid mass, and even at my skinniest adult weight, early in grad school when I was on a major health kick and running every day, I was just under 200 pounds and think that I was probably a bit too thin.  I am a person who relishes good food, however, and also tend to turn it for comfort, and by the time that I left MIT, I was up to 235 pounds and not so happy about it.

Then my scientific life intensified, and so did my stress and my amount of travel.  Travel is a killer, when it comes to weight, because travel means disrupted eating habits, restaurant dining, and free pastries, cookies, and other treats calling out tempting songs from the coffee breaks of every conference, every working meeting, and every program review.  Restaurants seem to soak everything, even every vegetable, in tasty fats, and I become bloated, weight climbing and energy falling.  As my career progresses, my stress and travel have both increased, and early last year I felt a shock of fear as I saw my weight brush up nearly to 260 pounds.  In that moment, I took a hard look at my sluggish feelings and fatigue, and I thought about my daughter, about the example I set for her and about the fact that I want to live a long, long life as her parent, and I decided that I would have to change my life.

It's been slow and hard and not without its setbacks, but I've been managing my weight much better. As always, there is no secret to it, just a consistent long-term increase of activity and a complementary decrease in my caloric intake.  For activity, my FitBit really helps me, because I am not the sort of person who does well with "heroic" gym time plans: my life is too variable and disrupted, and it works a lot better to just be adding a few more minutes walking here and there throughout the day, taking a circuitous route to get where I am walking, parking at the far end of the parking lot, etc.  For eating, I am a person for whom "eat less" is not a useful option, so I have focused instead on "eat lower caloric density" and have rediscovered in myself a love of many forms of vegetables: my snap peas, brussels sprouts, broccoli, asparagus, cherry tomatoes, carrots, etc. bring the strong tastes and umami satisfactions that can satisfy me and that fill my belly so that I need not suffer for my health.

But still, my travel remained my Achilles' heel, hurling great unhealthy caloric surges every few weeks: I could try to bring some vegetables with me, but without refrigeration I'd start to worry about their lifespan, and often it would take very little time for them to turn quite nasty.  And that's where having spent so much time thinking biologically over the past few years suddenly turned on a light in my mind and made me rediscover something that will no doubt be obvious to anyone who has spent time growing their own vegetables.

You see, vegetables, like us, prefer to live and thrive in a world of open air and oxygen. When we buy them, fresh vegetables are actually still quite alive, and in an open air environment they will try to stay that way for some time, resisting fermentation and rot, and continuing to fight the pathogens that would like to eat them.  My ancestors knew this well, when they stored their root vegetables in the cellar for the winter. When we package vegetables for storage in a refrigerator, though, we seal them in airtight bags, because in a refrigerator the cold slows down the metabolism of their cells and the biggest threat is not decay, but the dehydration of the refrigerator's cooling system.  Take them outside and leave them in their sealed package, though, and in the higher temperature and their enclosed state their respiration is inhibited, the water they emit begins to soak their environment, and they soon begin to spoil.

And so, my friends, once the problem had clearly reappeared within my sight, the solution was also obvious: before each trip, I open up every bag of vegetables, breaking their sterile seal.  The air comes in to help maintain their environment, and in return for this little bit of engineering of my vegetables, I find that they can nourish me in return for many more days.

Sufficiently happy vegetables and fruits, living on the desk in my hotel room.  The rubber bands do not fully close the openings, but keep the vegetables from falling out all over my bags when I travel.