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.)

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

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.