Jan 232009
ResearchBlogging.orgNumerous and diverse biological processes depend on the functioning of an internal clock. Biological timers determine your heart rate, the frequency of cell division, and the way you feel at 3 AM, among other things. Similarly, mechanical and electronic clocks serve essential functions in many kinds of man-made devices. As we begin to develop synthetic organisms for medical and industrial purposes, it will be useful for us to be able to construct timers within these micro-organisms to control their activity. In two recent papers, scientists have created molecular systems in mammalian and bacterial cells with tunable oscillation periods.

Although the methods used to construct these oscillators and the kinds of cells they were made in differ significantly, the two systems had one key similarity. Both oscillators used both a positive and a negative feedback loop. In principle, it should be possible to construct an oscillating system using only a negative feedback loop. For instance, you could have a system in which a transcriptional activator enhances the expression of a functional protein as well as that of a transcriptional repressor. As the concentration of the repressor increases, that of the activator falls, causing levels of the functional protein and the repressor to fall, allowing the concentration of the activator to rise again. By tuning the lag in this system one could in theory produce an oscillator with a range of possible frequencies. Yet many systems seem to have evolved with a positive feedback loop as well (in which the activator enhances its own expression).

This curious feature was the subject of a series of simulations reported by Tsai et al. (1) last July in Science. Their studies indicated that a system using only a negative feedback loop would produce a periodic oscillation just as expected. However, they also found that systems relying only on negative feedback were limited in that it was difficult to adjust the frequency of the oscillation without also altering its amplitude. Introducing a positive feedback loop stabilized the system so that the oscillator could be tuned to a wider range of frequencies without altering peak amplitude.

The benefits of this approach were demonstrated in data reported by Stricker et al. last November in Nature (2). They constructed a circuit that expressed green fluorescent protein in an oscillatory manner in response to stimulation by arabinose and isopropyl β-D-thiogalactopyranoside (IPTG). They created a circuit (see their figure, right) in which every component ran off a hybrid promoter that could be activated by AraC (which binds arabinose) and inhibited by LacI (which binds IPTG). Arabinose binds to and activates AraC, while IPTG binds to and inactivates the LacI repressor, with the result that all three genes are transcribed, and the cells fluoresce due to the presence of GFP. As the concentration of LacI increases, the activating power of the IPTG decreases, leading to an eventual repression of transcription and the end of fluorescence. As the LacI proteins get degraded the IPTG concentration again becomes sufficient to activate transcription, leading to a new fluorescent phase.

Stricker et al. found that they could alter the frequency and amplitude of this oscillation by altering the growth conditions of the bacteria (temperature and nutrient availability) as well as by adjusting the concentrations of the activating reagents arabinose and IPTG. By attempting to match computer models of their oscillator to the data they collected, they found that the time needed for translation, folding, and multimerization played a critical role in establishing the existence and period of the oscillation. Stricker et al. constructed an additional circuit using only negative feedback from LacI, proving that this was possible, but they found that in this case the period was not very sensitive to IPTG concentration and the oscillations were not as regular.

A similar system was constructed in Chinese hamster ovary cells by Tigges et al., who described their results recently in Nature (3). The circuit they constructed used tetracycline (TC) and pristinamycin I (PI) as activating molecules. The tetracycline-dependent transactivator (tTA) served as the positive feedback lood, activating transcription of itself, GFP, and the pristinamycin-dependent transactivator (PIT). In this system, increased levels of PIT cause the production of antisense RNA to tTA, causing its mRNA to be destroyed prior to protein production. This, in turn, diminishes production of all proteins until the reduced levels of PIT allow tTA to again activate transcription. They found that they could control the period of oscillation by altering the gene dosage (i.e. the quantity of DNA used to transfect the cells).

The oscillating systems constructed in these papers serve more as test cases and examinations of principles than as functional pieces of synthetic systems. You will not be using an E. coli alarm clock any time soon. However, it has always been true that you learn more from trying to build something than from trying to tear it apart. These attempts to construct artificial periodic oscillators have provided interesting insights into those that have evolved naturally. The knowledge gained from these experiments will help us to understand oscillatory systems like the circadian rhythm and cardiac pacemaker, in addition to illuminating design principles for synthetic biology.

(1)T. Y.-C. Tsai, Y. S. Choi, W. Ma, J. R. Pomerening, C. Tang, J. E. Ferrell (2008). Robust, Tunable Biological Oscillations from Interlinked Positive and Negative Feedback Loops Science, 321 (5885), 126-129 DOI: 10.1126/science.1156951

(2)Jesse Stricker, Scott Cookson, Matthew R. Bennett, William H. Mather, Lev S. Tsimring, Jeff Hasty (2008). A fast, robust and tunable synthetic gene oscillator Nature, 456 (7221), 516-519 DOI: 10.1038/nature07389

(3)Marcel Tigges, Tatiana T. Marquez-Lago, Jörg Stelling, Martin Fussenegger (2009). A tunable synthetic mammalian oscillator Nature, 457 (7227), 309-312 DOI: 10.1038/nature07616

 Posted by at 12:45 AM
Mar 252008
ResearchBlogging.orgBiological systems have the interesting property that most of the reactions enabling life processes are, when left to their own devices, exceedingly slow. To reach the timescales that we associate with “living”, these reactions must be sped up, which requires the presence of enzymes. Because they significantly enhance reaction rates under conditions that can be encountered almost anywhere, the design of artificial enzymes is an active area of research. In two papers this month, David Baker’s lab describes notable success in designing enzymes in silico to have specific activities with significant (106-fold) rate enhancement.

As a graduate student at UNC, I was fortunate to interact frequently with Richard Wolfenden, who did a great deal of work to find out just how good enzymes are at what they do (1). The fact is that there is a wide range of activities and rate enhancements. The proline isomerase cyclophilin, for instance, achieves a modest 106-fold rate increase, depending on the substrate. In contrast, arginine decarboxylase achieves an amazing rate enhancement of about 1019. Many reactions we think nothing of, such as hydrolysis of a phosphodiester bond (found in nucleic acids) would take millions of years in pure neutral water at 25° C. Of course, deviations from neutral pH and the presence of other molecules greatly enhance these rates, and obviously the same is true of changes in temperature, but this is a useful starting point for comparing enzymes to basal rates.

In the works at hand, collaborative teams involving several labs coordinated by David Baker designed enzymes to perform a novel retro-aldol reaction (2) and the Kemp elimination from 5-nitro-benzisoxazole (3) (a proton abstraction causing a ring to open). The retro-aldol paper is fascinating, particularly because of the multi-step nature of the reaction, but I’m going to focus on the Nature paper because its results are more complete, in that they implemented an appropriate wet-lab extension to the computational procedure.

The fundamental strategy of both papers is the same. For most enzymes it is believed that catalysis occurs because the transition state, the moment when the chemical reaction has the highest energy, is stabilized by the functional groups of the enzyme (see (1), among others). Using their knowledge of chemistry, the researchers of these groups predicted a transition state, and then positioned functional groups of side chains in such a way that they would stabilize this predicted state. They also placed potential bases in an appropriate geometry to attack protons as necessary. This done, they used a program based on Baker’s ROSETTA to predict sequences that would fold to produce this geometry. This required a somewhat more complicated process in the case of the retro-aldol reaction due to its multiple steps.

One interesting outcome was that TIM barrels were a popular choice of this algorithm in both papers. The final results in the Röthlisberger paper are all based on backbones identified by CATH as TIM-barrel folds (explore these scaffolds at the PDB: 1thf, 1a53, 1h61, 1jcl). As the authors note, the TIM barrel is a very common catalytic scaffold in nature, in part because the central β-strands provide a convenient way to orient side-chains towards the catalytic pocket. In both papers, the structures predicted using the ROSETTA algorithm were shown to be very close to the actual result, although they only checked successful catalysts. A comparison of the failed designs to their predicted structures may be of great use in refining the computational approach.

As the above paragraph implies, the groups did in fact succeed in designing enzymes that achieved significant rate enhancements. In the case of the Kemp elimination, the eight enzymes reported had ~5×103 – 2×105 -fold increases in rate over the spontaneous reaction in a very slightly basic solution. This amounted to actual kcat (reaction rate) values of 0.006 – 0.29 s-1, which is significantly slower than is common for enzymes.

In order to improve these results, Röthlisberger et al. turned to the process that produced our own prodigious enzymes in the first place, i.e. evolution. Using a relatively standard in vitro evolution approach, they altered one of the early successes, KE07, which had a kcat of 0.018 s-1. Keep in mind, this was not the best computational design result, just one of the first that worked. This in vitro evolution procedure, in just a few rounds, produced an enzyme with a kcat of 1.37 s-1. While this is still slow for an enzyme, it represents a rate enhancement of ~1×106 over the spontaneous reaction in solution, an acceleration comparable to that of a modest enzyme like cyclophilin.

This is nowhere near a complete journey. I’ve already mentioned that the enzymes produced in these experiments are still quite slow in comparison to the genuine article, and the rate enhancements are still modest. The specificity of the enzymes also has yet to be proven—can these proteins distinguish their targets from a sea of similar molecules, or are they promiscuous catalysts? A further dissection of the failed designs is essential to refining the computational approach employed. More careful consideration of effects beyond the secondary shell, and (as the authors note) backbone dynamics and loop positioning may prove particularly helpful in future iterations.

So, we are not all the way to the creation of a truly proficient man-made enzyme, but this is a tremendous step in that direction. The combination of wet lab and computational approaches proved to be very successful in this case. In principle, it should be possible to incorporate all that was learned in the in vitro evolution experiments into the design algorithm from the start. We will not be designing custom catalysts for biofuel production and bioremediation tomorrow or next week. These results, however, demonstrate substantial promise for the future.

In particular, the retro-aldol paper suggests that this approach will work for multi-step reactions. However, provided that the intermediates are stable and soluble this will not be strictly necessary. So long as efficient catalysts can be designed for each step, the ability of ROSETTA to design protein-protein interfaces will make it possible to assemble functional synthetic or catabolic enzyme cassettes to achieve very complex chemistry with tremendous accelerations over basal rates.

1. Wolfenden, R., Snider, M. (2001). The Depth of Chemical Time and the Power of Enzymes as Catalysts. Accounts of Chemical Research, 34 (12), 938-945. DOI: 10.1021/ar000058i

2. Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Rothlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F., Hilvert, D., Houk, K.N., Stoddard, B.L., Baker, D. (2008). De Novo Computational Design of Retro-Aldol Enzymes. Science, 319(5868), 1387-1391. DOI: 10.1126/science.1152692

3. Röthlisberger, D., Khersonsky, O., Wollacott, A.M., Jiang, L., DeChancie, J., Betker, J., Gallaher, J.L., Althoff, E.A., Zanghellini, A., Dym, O., Albeck, S., Houk, K.N., Tawfik, D.S., Baker, D. (2008). Kemp elimination catalysts by computational enzyme design. Nature DOI: 10.1038/nature06879

 Posted by at 1:30 AM
Mar 012008
ResearchBlogging.orgContinuing the banner day, Daniel G. Gibson and colleagues have published a rather subdued article in Science. In this truly impressive experiment, a group from the J. Craig Venter Institute synthesized and assembled an entire 583 kilobase-pair genome of Mycoplasma genitalium (1). Besides being a fairly cool piece of work all on its own, this outcome has profound implications for the future of bacterial research and the development of custom organisms

The stated purpose of the research is to investigate M. genitalium itself, which has the convenient property of possessing a very small genome. As the authors note (emphasis mine):
Approximately 100 of its 485 protein-coding genes are nonessential… it is not known which of these 100 genes are simultaneously dispensable. We proposed that one approach to this question would be to produce reduced genomes by chemical synthesis

One might reasonably conclude from this passage that JCVI has a dearth of small thinkers: this is akin to proposing to light your yard by shifting the position of the moon.

Yet Gibson et al. pulled it off using a tiered assembly method. They synthesized 101 overlapping pieces of DNA ~6 kb long. Using in vitro recombination they assembled groups of these into 24 kb fragments and propagated them using bacterial artificial chromosomes. Using a similar strategy they managed to continue assembling fragments up to about 144 kb in size; beyond this, however, the in vitro method did not work. So they turned to the yeast Saccharomyces cerevisiae to assemble the larger “C” fragments into half-genome and even whole-genome constructs. They then checked the whole genome for errors using the famous “shotgun” approach. Amazingly, their first run produced only two errors, both human in origin. After some wrangling they ironed it all out and ended up with precisely the genome they wanted, complete with special “watermarks”.

So they managed to construct an enormous hunk of DNA. What’s the benefit? Well, they can do those crazy experiments that they’re planning, for one thing. For another, assuming they can figure out how a mycoplasma works, they can design a new mycoplasma that does something special. For instance, they could design a new genome containing genes that allow the bacterium to eat toxic materials, or purify uranium out of the soil, or turn biomass into octane.

Granted, it is possible to get some of these genes into a bacterium using plasmids or BACs. However, what goes in can also go out—ejecting a plasmid is relatively easy, especially if it doesn’t provide any selective advantage. Completely ejecting a genomic cassette, while possible, is much more rare. Moreover, if you can custom-design the genome, then the custom enzymes can be interspersed with essential genes, making deletion that much more unlikely. Thus, once the genome can be manipulated at will, the only limitation on what an organism can be made to do is the extent of our ability to find or design proteins that fulfill the desired function. Figuring out which of a hundred-odd genes from M. genitalium can be done away with is just an efficiency step to make space for new genes to do whatever we want.

This approach will be a powerful tool for other experiments as well, don’t get me wrong. For instance, you could use the approach to place custom tags around certain genes so that they get excised at particular times or in a conditional manner, or you could swap sequences around to get a better idea how important gene arrangement is. But there’s no denying the enormous advance that this success makes possible, despite the authors’ decision not to speculate in the paper. The genome-synthesis technique is an essential step in the construction of a completely custom organism.

1. Gibson, D.G., Benders, G.A., Andrews-Pfannkoch, C., Denisova, E.A., Baden-Tillson, H., Zaveri, J., Stockwell, T.B., Brownley, A., Thomas, D.W., Algire, M.A., Merryman, C., Young, L., Noskov, V.N., Glass, J.I., Venter, J.C., Hutchison, C.A., Smith, H.O. (2008). Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome. Science, 319(5867), 1215-1220. DOI: 10.1126/science.1151721

 Posted by at 2:20 AM