Monday, October 11, 2021

Meeting Measurement Precision Requirements for Effective Engineering of Genetic Regulatory Networks

We've got a new preprint up today, "Meeting Measurement Precision Requirements for Effective Engineering of Genetic Regulatory Networks", that is an unusual mixture of theoretical analysis and interlaboratory study. 

The work started out as an investigation of the replicability of flow cytometry measurements. Flow cytometry, as readers of this blog may know, is one of my favorite biological measurement tools, since it lets us obtain measurements from large numbers of individual cells. I've been involved in a number of projects that have put it to good use in engineering biological devices, and the calibration methods available let us put real, biologically-sensible units on the measurements. But just how good are these measurements and how reproducible?  That's what we set out to study with a consortium of collaborators and about two dozen flow cytometers.

Then we went to go write it up, and a rabbit hole opened beneath our feet, sucking us down into an unexpected set of theoretical questions. We had a number (~1.5-fold precision), but was that a good number? In fact, how do we even decide what a good number is? What do you even need to do good engineering? 

Maybe we should have just called that "future work" and published what we had. But we didn't. We followed that rabbit hole down and the manuscript went into limbo. But when it came out of limbo, the manuscript was standing on its head and had an answer. What started as an investigation of flow cytometry became an investigation of the general requirements for effective biological engineering, with the work on flow cytometry becoming one verified answer for how to meet those requirements.

Basically, you want to be on the left side of the red line.

We ended up with a (highly abstract, conservative) formula for estimating how well one needs to know values in order to engineer gene regulation. And for most state of the art work, it means you need to have a measurement precision somewhere in the range of 1.2-fold to 2.0-fold, with calibrated flow cytometry right smack in the middle.

I'm happy with these dual results, and I think they should be useful to help us move another couple of steps towards a world of reliable and predictable biological engineering.

Thursday, July 15, 2021

Predictable signal amplification with recombinases

New paper out today: "Quantitative characterization of recombinase-based digitizer circuits enables predictable amplification of biological signals." If we ever want to be able to make reliable controller in cells, we need to have well-separated control signals. Many of the biological sensors and other inputs that we work with, however, are really blurry, so we need devices that can clean them up. This paper demonstrates how this can be done in mammalian cells with a circuit that cleans up a poorly separated signal by nearly 3 decibels!

Blurry input (left) is predicted to be separated well by our recombinase device (middle), and that prediction is realized experimentally (right).

This work, part of the NSF Living Computing Project,  involved collaboration across several labs and a lot of work to connect the devices, analytics, and models. Making this work meant really getting down into what we wanted not just biologically but computationally, in terms of the signal properties of the device. The models and metrics guided adjustments in device design that ultimately feed back into a better performing system. I'm personally very happy with the result as an example of a getting really serious about the engineering approach biological systems.

Monday, June 07, 2021

From reproducibility failure to methodological success

Out today in PLOS ONE, "Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs" solves a mystery hiding in the iGEM interlaboratory studies for 2016, 2017, and 2018. You see, the publication of the 2017 interlab data was delayed, even after the publication of the iGEM 2016 study and iGEM 2018 study, because of a troubling mystery: the plate reader results from the 2016 and 2017 studies did not match.  This was a shock, because the 2017 study was intended to be a replication of the 2016 study, plus a few extensions and enhancements. But what we got was shockingly different, systematically off by a factor of more than 10. So which, if either of them, was right?

This is a terrible and unsettling place to find oneself in, but we couldn't actually answer the question until after we had run and analyzed the 2018 study. With that study, we finally had a way to put plate reader data on the same scale as flow cytometry data, so that we could assess accuracy through two independent measurements. So once we'd finally finished analyzing and publishing that data, we turned to comparing the three years to find out what had happened and how to understanding our failure to reproduce. And here is the story, finally, summed up in a single image:

It appears the 2017 plate reader results were right: they match both the 2018 results as well as the flow cytometry from 2016. There's a lot more detail in the paper, as well as additional confirmations, but the bottom line is that it looks like the calibrant that we prepared for the 2016 study did not have the concentration of fluorescein that it was intended to. 

Embarrassing, but actually, I think, good news in the end. Because we could tell! We are no longer held to the tyranny of uncertainty, unable to even know if our measurements have been reproduced. With multiple independent measures and a successful confirmation of values reproduced in three different studies (2016 flow, 2017 plate, 2018 both), we now have truly solid ground on which to stand, biologically. Every future study that we build can bootstrap off of these results, and know if the numbers that come out are reasonable or not.

But why are we still preparing our own fluorescent calibrants in the first place? We need metrological traceability and easily purchased commercial preparations with adequate quality control, just like we have for units of time and length. Calling all reagent suppliers: who will first start to sell a plate reader cellular quantification kit?

Monday, May 31, 2021

Analysis and Visualization of Gene Expression Data

A couple of days ago, I gave a seminar on analysis and visualization of gene expression data for After iGEM, which was recorded and made freely available online. The first half of the talk is focused on core issues on data analysis, covering unit calibration, use of geometric statistics, process controls, and relating measurements to biology. The second half is about how to make a good figure, applying lessons from my favorite instructor in the area, Edward Tufte, that are likely useful to anyone and everyone who makes a figure ever. For those interested, I'm embedding the video below, and have posted the slides on my website.

Monday, April 05, 2021

Sharing our ignorance

One of the both wonderful and challenging things about working in a highly interdisciplinary area like synthetic biology is that all of us who work there are painfully ignorant. 

No matter how much of an expert one is in some areas, there is simply too much complexity and too many things to know to allow one to be an expert in all of the relevant aspects of the field. Even an apparently simple task like measuring fluorescence from simple genetic constructs often contains quite a number of rabbit holes of complexity that one can go down. 

Working in a field like this, it's easy to feel insecure about how much one doesn't know. But ignorance can be a gift as well, providing an outside perspective and shedding light on unexamined assumptions. Moreover, what is collaboration if not constructive use of complementary ignorance? Indeed, this is what Joy's law is all about: tackling complex challenges is effectively impossible for "lone geniuses" and always involves expertise dispersed among many different people.

This is a key part of what we are trying to address with the Synthetic Biology StackExchange proposal. Knowledge flows slowly and noisily through person-to-person networking, but much more quickly through well-curated community Q&A like StackExchange supports. Instead of one person getting their question answered through oral tradition, we all get an answer that's confirmed by many peer reviewers and made easy to find for the next several hundred people who need to know.

All we need now to make this happen is another few dozen people to support the proposal and then come ask three good questions on one of the existing sites like Biology.SE or Bioinformatics.SE (thus hitting the "people able to use StackExchange" criteria for launch).  I've really been enjoying this myself, asking questions about simple laboratory information that's outside of my experience, like how hard it is to pipette right and the shelf-life of frozen bacteria, and receiving interesting and informative answers.

Come join us today and make a gift of your ignorance!

Friday, March 12, 2021

One year of lockdown

One year ago today, I was in England, nervously finishing up a standards meeting and hoping that I could make safely home without either getting infected with Covid or getting stuck on the wrong side of the border. 

On March 13th, 2020, I flew home via Chicago, on the last day before the border closed. My wife and I embraced, and we put our household into lockdown. We waited nervously for a week of potential incubation time, but I had apparently escaped infection. Spring break began, and we wondered if there would still be school at the end of it.

We were fortunate. I had noticed the potential trouble building and we had at least a month's supplies laid in our basement for our household. My wife and I could both keep doing our jobs online, and despite some friction with the kids and cats, we settled into an enclosed routine. I miss being able to get together with friends in person, but between my work and family, my life is full and over-full with social interactions, and in the evenings I usually just want a quiet place of solace. 

Some things, I am surprised that I do not miss, like restaurants. We've gotten much better at cooking, and the food we eat is healthier. I've lost fifteen pounds or so, thanks to my healthier lifestyle. More time with the kids is a silver lining too. But what a world of change we've been through, all of us, and it's not over yet.

Tomorrow will be the anniversary, one full year since the last time our house was open to the world. One full year of living our pandemic lives. 

There's a light at the end of the tunnel now, I think, but we're a long way yet from done. Our indoor cats perch on the windowsill, looking at the world outside denied to them to roam, and I think I can identify.

Thursday, March 11, 2021

Building the SynBio StackExchange community

We're getting closer to launching the Synthetic Biology StackExchange Q&A site, but building a new community on StackExchange is hard, and we need more people to come help!

There are more than 150 different topic sites in the StackExchange network, and over the course of launching them, StackExchange has learned a lot about what causes a community to succeed or fail. Most important, it seems, is having critical mass at the start, and they've set the thresholds for site launch accordingly.

What this means for SynBio StackExchange is that we'll launch as soon as we have:

  • 250 people committing to use the site when it launches, and
  • at least 100 of those having 200+ reputation on some other StackExchange site.

I've believe that both of these are quite reasonable and achievable. The 250 people threshold is pretty straightforward, and we're on track to hit that around the end of March. The second criteria, however, is harder since most SynBio folks aren't yet contributing to StackExchange, and will be the one that determines when we can actually launch the site.

Getting 200 reputation is pretty easy: it only takes about 4 questions or 3 answers. It's a big deal, though, since it means you've put a little skin in the game and figured out how to contribute on StackExchange. And once you learn to play the game, it's often pretty fun: helping people feels good, getting your own problems solved is great, and StackExchange is set up to really reward people who put in just a few minutes on a regular basis.

And so it is with building this community. Come join us, and bring one friend. Ask another one tomorrow, and ask your friend to do the same. We're doing pre-launch Q&A using the Biology StackExchange synthetic biology tag, so come ask one question there. Then ask another one tomorrow. And if you want more, ask me for an invitation to the community-building Slack where we're sharing tips and helping each other.

One step at a time, we're working to build this community, and when it's big enough, we'll get to have a dedicated watering hole where everybody who needs help in synthetic biology knows that they can come and find it.

Thursday, February 25, 2021

Getting close to a SynBio StackExchange!

Synthetic Biology StackExchange is one step closer to launching! We've now passed the definition phase for the site, and as soon as we have a critical mass of people committing to participate in the beta, the site will be officially launched. 

I'm very excited about getting this site going, since I think it will be an extremely valuable resource for many thousands of folks working in the area. I know even as an expert, I'm quite naive about a lot of details in the laboratory and issues outside of my own areas of expertise, and really look forward to asking as well as answering questions.

If you're interested too, please go to the site and commit to the beta today!

Monday, February 08, 2021

Come help define a Synthetic Biology StackExchange!

In the actual practice of working in synthetic biology, there are so many pragmatic details that don't get captured well in scientific papers. Right now, they are basically passed around by word of mouth, through oral tradition amongst students in a lab or hallway conversations at conferences. But there is a better way.

Pretty much everybody who programs makes use of StackOverflow, which provides well-curated answers for programming questions. The greater network of StackExchange sites spun off of it provides a great one-stop-shop for lots of other communities as well, from math, physics, and chemistry to travel, cooking, and personal finance. The iGEM Engineering Committee, as part of its educational mission, is trying to do the same for synthetic biology

Synthetic biology field is rapidly growing, highly cross-disciplinary (which means none of us can be experts at everything), and we think it could use a good, universal database of questions and answers. It will be good for students learning the field, and also good for professionals who need to know things outside of their personal expertise.  

Right now, we're in the "definition" phase at StackExchange, and need about 100 more people to add example questions and vote for example questions they like. Just follow this link or click on the imagebelow, make an account, and you can start asking and voting too!

Sunday, January 24, 2021

From art to engineering in synthetic biology

 Two weeks ago, I gave a talk called "From art to engineering in synthetic biology" at the Build-a-Cell seminar series. This talk pulls together a lot of different threads of research that I've been working on, and there were some really enjoyable questions to answer afterwards, and Build-a-Cell has put the video recording of it up online, so if you're interested I hope you will enjoy and share!