This semester, I've been mentoring a team of two freshmen working on designing a cooperative system of biofuel-producing algae and nutrient-recycling bacteria. It's a really neat project -- in fact all three of this year's 20.20 projects are really interesting. They have more of a focus on system dynamics / population engineering, where my year we focused on devices. I think this may be due to having Ron Weiss instead of Drew Endy. (Another indication of Weissitude is that we're all modeling our systems in MATLAB... sigh. I am not fond of MATLAB.)
I don't actually know much about biofuels, synthetic cooperation, or complex metabolism. I also don't know much about mentoring a team in the sort of hands-on, hands-off style that's appropriate when the point is for everyone to learn and stretch themselves. So it's a challenge, but an extremely satisfying one. I have watched my team drink the synthetic biology Kool-aid in the most remarkable way. There was a palpable transition from naivete to relative understanding;
Mentorship is important, but providing it at this level is also exhausting. Every day after class I feel physically tired (actually, that may have more to do with my sleep schedule than my mentoring schedule).
Showing posts with label class:20.20-synthbio. Show all posts
Showing posts with label class:20.20-synthbio. Show all posts
Monday, April 19, 2010
Sunday, March 21, 2010
Journal Club Followup: The amber-suppressing AND gate
We went over the AND gate paper in class, and generated a lot of constructive criticism that I hadn't thought of by just reading it on my own. I guess this is what class is for, huh.
In particular, we addressed the question of whether the AND gate is truly modular. As I discussed in the previous post, in principle this AND gate is modular in the sense that it can be plugged into different inputs and outputs. However, this isn't the whole story. "Plugging it in" to a different input is not so simple as just putting a new promoter in front of Input Gene 1. The whole promoter/RBS/coding-region assembly has to be tuned to have an appropriate strength. If you swap in a stronger promoter, you might have to weaken the RBS (ribosome binding site) in order to get just the right amount of protein expression to feed in to the rest of the AND gate.
In fact, that's exactly what the authors had to do when they first put their AND gate together, which I kind of glossed over. Recall that the system works by expressing an mRNA with amber stop codons in, and the amber-suppressor tRNA that can read those stop codons; the mRNA codes for a protein that transcribes the output promoter. Now, in principle this should Just Work. But in practice, if there's enough mRNA around, you can get spontaneous read-through even if the amber-suppressor tRNA is supposedly turned "off", for two reasons. One, even if the tRNA is "off", it might still be produced at a low basal level. Two, the amber stop codon is relatively "weak", and sometimes just gets read through anyway. (That is, it's not very good at recruiting the translation-stopping machinery, which is supposed to disassemble the ribosome and cut loose the newly translated protein.) So the authors had to adjust the RBS that governed translation of the mRNA, so there wouldn't be too much of it floating around and you wouldn't get this spurious effect.
Apparently this sort of adjustment is called "impedance matching", for those of you who are more familiar with electronics than cells. I don't know enough about electronics to explain exactly what impedance matching is or why it's a suitable analogy here, but it boils down to "make sure all the wires are carrying appropriate amounts of current, and if you connect something new to a wire you might have to add a resistor or something to fix the current back to how it used to be."
So, no, it's not plug-and-play quite yet. More like plug, mutagenize the RBS, and play... but maybe we'll get there eventually.
The other main criticism of this AND gate, from a modularity point of view, is that you can't have two copies of the gate in the same cell and expect them to operate independently. mRNAs and tRNAs float around, and if one AND gate is expressing mRNA and the other is expressing tRNA, then both of them will output ON, even though both of them ought to be OFF. This is a more serious problem, because you can't just tune an RBS and expect it to go away -- this design for an AND gate is in principle not modular with respect to other AND gates placed in the same cell. (Possible workarounds include hiding different AND gates in different cells and mixing several populations together, but then you have to work with cell-to-cell cooperation, which is a whole different ballgame.)
In particular, we addressed the question of whether the AND gate is truly modular. As I discussed in the previous post, in principle this AND gate is modular in the sense that it can be plugged into different inputs and outputs. However, this isn't the whole story. "Plugging it in" to a different input is not so simple as just putting a new promoter in front of Input Gene 1. The whole promoter/RBS/coding-region assembly has to be tuned to have an appropriate strength. If you swap in a stronger promoter, you might have to weaken the RBS (ribosome binding site) in order to get just the right amount of protein expression to feed in to the rest of the AND gate.
In fact, that's exactly what the authors had to do when they first put their AND gate together, which I kind of glossed over. Recall that the system works by expressing an mRNA with amber stop codons in, and the amber-suppressor tRNA that can read those stop codons; the mRNA codes for a protein that transcribes the output promoter. Now, in principle this should Just Work. But in practice, if there's enough mRNA around, you can get spontaneous read-through even if the amber-suppressor tRNA is supposedly turned "off", for two reasons. One, even if the tRNA is "off", it might still be produced at a low basal level. Two, the amber stop codon is relatively "weak", and sometimes just gets read through anyway. (That is, it's not very good at recruiting the translation-stopping machinery, which is supposed to disassemble the ribosome and cut loose the newly translated protein.) So the authors had to adjust the RBS that governed translation of the mRNA, so there wouldn't be too much of it floating around and you wouldn't get this spurious effect.
Apparently this sort of adjustment is called "impedance matching", for those of you who are more familiar with electronics than cells. I don't know enough about electronics to explain exactly what impedance matching is or why it's a suitable analogy here, but it boils down to "make sure all the wires are carrying appropriate amounts of current, and if you connect something new to a wire you might have to add a resistor or something to fix the current back to how it used to be."
So, no, it's not plug-and-play quite yet. More like plug, mutagenize the RBS, and play... but maybe we'll get there eventually.
The other main criticism of this AND gate, from a modularity point of view, is that you can't have two copies of the gate in the same cell and expect them to operate independently. mRNAs and tRNAs float around, and if one AND gate is expressing mRNA and the other is expressing tRNA, then both of them will output ON, even though both of them ought to be OFF. This is a more serious problem, because you can't just tune an RBS and expect it to go away -- this design for an AND gate is in principle not modular with respect to other AND gates placed in the same cell. (Possible workarounds include hiding different AND gates in different cells and mixing several populations together, but then you have to work with cell-to-cell cooperation, which is a whole different ballgame.)
Saturday, March 6, 2010
The answer to my question about scaffolds
I passed my scaffold question on to my 20.20 professors, and got a very thoughtful response. (To their credit, it was also a very timely response; I've just been slow in posting it.)
Recall my question: there seemed to be a conflict between the role of kinase cascades as signal amplifiers, and the role of scaffolds as giving specificity. Turns out, naturally, that there isn't really a conflict at all. Rather, I didn't fully understand what scaffolds do. The crux of what I wasn't understanding is that proteins go on and off their scaffold all the time -- they don't stay bound (semi)permanently.
One of the main benefits of scaffolds is to make signaling pathways more efficient: they help bring proteins close together so they can interact. If you have only a few molecules of Protein A and Protein B, then they may not encounter each other very often if they're just freely diffusing around the cell. A scaffold can grab one Protein A and one Protein B, hold them close together long enough to interact, and the release them and go look for two new proteins. Or if A is a kinase for B, then A can stay on the scaffold and multiple copies of B can take turns.
Scaffolds also act like little switchboards, dictating where signaling pathways are allowed to cross and merge or where they must stay separate. Suppose a scaffold has one binding site for the kinase A, and a second binding site that can fit either Protein B or C, but not D. In this case you get activation of both B and C, and the signal from A propagates in two different directions, provoking two separate responses (but not provoking a third response). This is really helpful because a lot of kinases are promiscuous -- they'll phosphorylate anything they can get their hands on -- so if they're confined to scaffolds, then they'll do whatever the scaffold says they can do, and no more.
Recall my question: there seemed to be a conflict between the role of kinase cascades as signal amplifiers, and the role of scaffolds as giving specificity. Turns out, naturally, that there isn't really a conflict at all. Rather, I didn't fully understand what scaffolds do. The crux of what I wasn't understanding is that proteins go on and off their scaffold all the time -- they don't stay bound (semi)permanently.
One of the main benefits of scaffolds is to make signaling pathways more efficient: they help bring proteins close together so they can interact. If you have only a few molecules of Protein A and Protein B, then they may not encounter each other very often if they're just freely diffusing around the cell. A scaffold can grab one Protein A and one Protein B, hold them close together long enough to interact, and the release them and go look for two new proteins. Or if A is a kinase for B, then A can stay on the scaffold and multiple copies of B can take turns.
Scaffolds also act like little switchboards, dictating where signaling pathways are allowed to cross and merge or where they must stay separate. Suppose a scaffold has one binding site for the kinase A, and a second binding site that can fit either Protein B or C, but not D. In this case you get activation of both B and C, and the signal from A propagates in two different directions, provoking two separate responses (but not provoking a third response). This is really helpful because a lot of kinases are promiscuous -- they'll phosphorylate anything they can get their hands on -- so if they're confined to scaffolds, then they'll do whatever the scaffold says they can do, and no more.
Monday, March 1, 2010
Ideas dump
I'm looking over some old brainstorms, because of course I'm not busy at all, no sir, ahahaha... Anyway. I keep getting neat ideas, and I'm going to put some of them here so that (1) I don't forget them before I find time to investigate and (2) maybe someone will actually look at them and think about them. Also, I apologize for not explaining these ideas for the benefit of biology neophytes.
- Enzymes that bind DNA and perform actions including cutting/pasting DNA or recruiting other enzymes. Can you inhibit their action by adding short pieces of RNA (or similar) with the same sequence as their binding site?
- RNAi seems to exist in bacteria, kind of (not as much as in C.elegans). Is it useful for creating synthbiological devices? I don't recall seeing any; what's the roadblock?
- In particular, RNAi could be a neat way of getting around the crosstalk/specificity problem, since it's very sequence-specific and designing RNA sequences is easier than designing proteins.
- What would it take to drive localization to a synthetic organelle?
- Need to learn more about riboswitches. All the riboswitches I've seen so far are the kind that respond to a small molecule. Hmm: can you make a riboswitch that responds to a short ssRNA??
- What are the primitives of biological circuits? In a regime where it's easier to build monolithic black boxes than to reuse parts, how do things like two-component signaling evolve, that almost look intelligently designed with modularity etc?
- I think I'm on an RNA kick. Is this justified?
- Whoa, DNA scaffolds? Clever! read this at some point
Monday, February 15, 2010
Journal Club: The amber-suppressing AND gate
Anderson JC, Voigt CA, Arkin AP. Environmental signal integration by a modular AND gate. Molecular Systems Biology 3:133 (2007) | doi:10.1038/msb4100173
Logic gates (AND, OR, NOT, etc.) are the basis of electronic computation. If we'd like to implement biological computation, one of our first steps has to be implementing similar logic gates using proteins and DNA. That is, we need to make devices that accept a few inputs, perform a logical operation on them, and then spit out the result. In the case of an AND gate, we want the output to be ON whenever both of the inputs are ON, and OFF when either input is OFF. It seems easy, but of course, this turns out to be a lot harder in biology (hence, people writing papers about it).
Figure 1: The result of this paper, if you abstract away all the interesting stuff. [Source]
What makes it hard to make an AND gate out of biochemical parts?
Lack of standard connectors. In electronics, every signal is carried by a current, and every connector is a wire. That isn't the case in biology. Biological signals are typically carried by the presence or absence of some protein that carries out some particular chemical reaction that affects other proteins. This is wildly nonstandardized, and it means if Protein A interacts with Receptor A, you can't just plug in Receptor B and expect things to work.
Fuzzy, non-discrete behavior. It's nearly impossible for a biological system to have a perfect ON or OFF state. Even if a signal is mostly off, there'll be a few molecules of it floating around somewhere. And when you go to turn it on, it'll take time. Basically, biological things tend to vary continuously and not discretely (in large jumps).
Crosstalk. If a biological device relies on some particular molecule, then that molecule is going to be everywhere in the cell. So, you can't put two copies of the same device into a cell and expect them to operate independently. They'll interfere or "crosstalk" with each other in ways you don't expect. In contrast, you can throw down dozens of electronic circuit elements onto a breadboard and they won't interfere with each other because they're separated by physical space. In biology, everything's in the same soup.
Logic gates (AND, OR, NOT, etc.) are the basis of electronic computation. If we'd like to implement biological computation, one of our first steps has to be implementing similar logic gates using proteins and DNA. That is, we need to make devices that accept a few inputs, perform a logical operation on them, and then spit out the result. In the case of an AND gate, we want the output to be ON whenever both of the inputs are ON, and OFF when either input is OFF. It seems easy, but of course, this turns out to be a lot harder in biology (hence, people writing papers about it).
Figure 1: The result of this paper, if you abstract away all the interesting stuff. [Source]What makes it hard to make an AND gate out of biochemical parts?
Lack of standard connectors. In electronics, every signal is carried by a current, and every connector is a wire. That isn't the case in biology. Biological signals are typically carried by the presence or absence of some protein that carries out some particular chemical reaction that affects other proteins. This is wildly nonstandardized, and it means if Protein A interacts with Receptor A, you can't just plug in Receptor B and expect things to work.
Fuzzy, non-discrete behavior. It's nearly impossible for a biological system to have a perfect ON or OFF state. Even if a signal is mostly off, there'll be a few molecules of it floating around somewhere. And when you go to turn it on, it'll take time. Basically, biological things tend to vary continuously and not discretely (in large jumps).
Crosstalk. If a biological device relies on some particular molecule, then that molecule is going to be everywhere in the cell. So, you can't put two copies of the same device into a cell and expect them to operate independently. They'll interfere or "crosstalk" with each other in ways you don't expect. In contrast, you can throw down dozens of electronic circuit elements onto a breadboard and they won't interfere with each other because they're separated by physical space. In biology, everything's in the same soup.
Monday, February 8, 2010
Courses for this semester
This semester's coursework has two themes: "Let's Read And Discuss Primary Literature", and "Time To Stop Abstracting Away The Physical Nature Of Biology". Both of my required bioengineering core classes are about cells, molecules, and tissues from a mechanical engineer's perspective. All three of my electives are literature-discussion classes on various topics.
I plan to blog a lot more about the papers I read in these classes. It probably won't be a full Journal Club post for every paper because I'll be reading about 6-8 papers a week, but I'll at least try to summarize them. (This is partly for your benefit and partly for mine -- I expect that writing paper summaries for the blog will help me read and understand the papers better.) But I will do full Journal Club posts for the papers that I find most interesting, and definitely for the ones I present. I might also write about interesting things that happen in the other two classes.
- 20.310 Molecular, Cellular, and Tissue Biomechanics - think introductory mechanics, only all the examples are biological things instead of, you know, steel beams or something. Up till now, I've been accustomed to thinking of DNA as a string of digital (AGCT) information, or maybe as a helical molecule; now it's time to think of DNA as a charged elastic rod, or a randomly-walking polymer. How much force can a motor protein exert to pull a vesicle where it's going? What happens when you push and pull on the cytoskeleton? What's the effect of pressure on cells? How do bones reshape themselves in response to forces?
- 20.330 Fields, Forces, and Flows in Biological Systems - fluids and E&M, only again all the examples are biological things. How do things like diffusion and electrophoresis work? How can you model the cell membrane as an RC circuit? What's the best shape / flow pattern for this sample chamber so that the most protein binds to the sensor on one side?
- 20.385 Advanced Topics in Synthetic Biology - this is paired with a freshman design/seminar course, 20.20, which I took two years ago and which rocked my world so hard. It's a great introduction to synthetic biology. The frosh get to do design projects, and because it's surprisingly hard to do this when you've only had introductory biology, the upperclassmen mentor the frosh teams. And when we're not busy mentoring, we have synthetic biology journal club. I'll be presenting a couple of papers and I'm super pumped.
- 7.25 Biological Regulatory Mechanisms - this just sounds fascinating. All the different ways gene expression or protein action can be controlled. Apart from being cool, this is also highly relevant for synthetic biology. Even apart from that, I'm excited about the lectures. This class involves picking apart the experimental logic of papers in a more rigorous way than I've ever had before, which I'm sure will be good for me as well as being fun. We're focusing on demonstrating results and excluding alternative explanations to an extent that seems to be missing in the modern age of "let's generate a zillion data points and then sift through them". Plus, it's a chance to pick the brains of some aged, sage professors. All in all, it really reminds me of ((my interpretation of) what Raffi said about) learning Talmud.
- 7.346 RNAi: A Revolution in Biology and Therapeutics - yet another paper reading class. I know nothing at all about RNA interference, but it's extremely important both theoretically and (potentially) medically. I'm also interested in using it to make synthetic-biological parts that don't crosstalk as much as protein-based parts do; we'll see if that's feasible.
I plan to blog a lot more about the papers I read in these classes. It probably won't be a full Journal Club post for every paper because I'll be reading about 6-8 papers a week, but I'll at least try to summarize them. (This is partly for your benefit and partly for mine -- I expect that writing paper summaries for the blog will help me read and understand the papers better.) But I will do full Journal Club posts for the papers that I find most interesting, and definitely for the ones I present. I might also write about interesting things that happen in the other two classes.
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