Synthetic gene circuits with a memory!

Imagine having a USB port in the body that we could use to insert a "flash drive" and transfer genetic data, therapies, or monitoring devices. The flash drive would have to be some kind of removable biological entity that has no problem getting in and out of the body. If you think about it, bacteria are the perfect candidates to be such devices. So, what if bacteria could be used as storage for genetic memory?

This is not so far-fetched if you think that recent studies have shown for example that genes expressed by bacteria in our guts can affect our propensity to be lean or fat. Bacteria have genes that "record" and "affect" what's going on in our body. The question is: can we control them?

Bacteria have a way of turning "on" or "off" their genes based on stimuli from the environment. Synthetic biology studies ways of using these "switches" to make "gene circuits". Genetic regulatory circuits are the biological analog of electric circuits, where genes, instead of light bulbs, are being turned on or off (by activating other genes).

Genetic circuits have numerous applications in medicine. For example, Auslaender et al. [1] used synthetic biology to create a pH sensor for cells. The researchers then implanted these cells into mice and used it as a device to detect diabetes. Lack of insulin causes an excess of acidity in the blood, and the pH drops below 7.35. Changes in pH induced by diabetes were quickly detected by the pH-sensor cells in the implanted mice. The pH information was processed and triggered a transgene expression response that resulted in the secretion of alkaline phosphatase to counteract the acidity. Basically, what the cells were able to do in the mice is: (1) detect the drop in pH; (2) trigger a response to restore the pH to normal levels.

In an electrical circuit you assemble elements like resistance and capacity. In a genetic circuit you assemble genes and "operators" able to edit the DNA in order to activate or deactivate the genes. One of such "editors" is a class of enzymes called recombinases. Apparently, there aren't many of these enzymes available, which limits the number of gene circuits one can make.

A recent study published in Science [2], however, presented a new class of such enzymes, derived from the bacteriophage Lambda, which is a virus that infects Escherichia coli. The novelty of the method doesn't stop here. You see, the goal is not just to have a working circuit, but to also make it autonomous. In other words, ideally, one wants a system able to detect responses and readjust the output based on the input it receives. The researchers devised genetic regulatory circuits able to "write", "input" and "read" genetic information.

Farzadfard and Lu [2] "converted genomic DNA into a 'tape recorder' for memorizing information in living cell populations." Their circuit, named SCRIBE (Synthetic Cellular Recorders Integrating Biological Events), responds to gene regulatory signals by generating single-stranded (ssDNA). The ssDNA is then coexpressed with a recombinase and introduces specific mutations in targeted positions of the cell DNA. The fraction of cells in the bacterial culture that carry the introduced mutations represents the biological memory at the population level.

For example, when the researchers exposed the cultures to an exposure input for 12 days (the equivalent of 120 generations in the bacterial population), they found that the

"frequency of mutants in these populations was linearly related to the total exposure time. Furthermore, we demonstrate that SCRIBE-induced mutations can be written and erased and can be used to record multiple inputs across the distributed genomic DNA of bacterial populations [2]."

It's a 'collective memory' embedded in the observed frequency of the mutation in the bacterial population. And the applications are almost infinite. I truly can't wait to see where this kind of research will take us in the future.

[1] Ausländer D, Ausländer S, Charpin-El Hamri G, Sedlmayer F, Müller M, Frey O, Hierlemann A, Stelling J, & Fussenegger M (2014). A synthetic multifunctional mammalian pH sensor and CO2 transgene-control device. Molecular cell, 55 (3), 397-408 PMID: 25018017

[2] Farzadfard, F., & Lu, T. (2014). Genomically encoded analog memory with precise in vivo DNA writing in living cell populations Science, 346 (6211), 1256272-1256272 DOI: 10.1126/science.1256272

Pumping fuel from bacteria

In my last post I discussed a bioengineered E. coli strain capable of producing an engine compatible biofuel. I hailed the finding as more efficient than ordinary biofuels because this technique has less environmental impact than biofuels from crops, for example, or cellulose, which instead use great amounts of water and forest land.

I did some more reading on the topic and found out that, surprise surprise, there are some costs in harvesting biofuels from bacteria as well, so my discussion was incomplete. However, there are good news at the horizon.

When I first read the Howard et al. paper, I imagined a petri dish of E. coli sitting in a slime of oil-like substance. I think I got confused with making yogurt. :-) In reality, the biofuel molecules are stored inside the cells (bacteria, in this case) and need to be taken out without harming the cells. Biofuel secretion strategies have been dubbed "milking." The difference, though, is that contrary to milk and cows, biofuels are generally toxic to the bacteria that produce them.

Several methods have been investigated to efficiently "milk" biofuel molecules out of bacteria without harming them. To understand these strategies, we need to learn a new concept: an efflux pump is a membrane transporter protein that carries a substance toxic to the cell outside the cell itself. These proteins remove all kinds of toxic substances, including antibiotics, for example, and they may be specific to one in particular, or carry a whole range.

In [1], Dunlop et al. discuss the use of efflux pumps in "milking" biofuels out of bacteria and reduce their toxicity to the cells:

"Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity."

The researchers first looked at the whole genome of E. coli to identify all genes encoding efflux pumps. They found 43 different pumps expressed in the E. coli genome, and tested them against a range of possible biofuels. Their strategy was as follows: the grew a culture of pooled bacteria with different subpopluations, each subpopulation expressing a different pump. In the absence of toxic biofuel-like substances, all subpopulations grew in equal proportions, and none had an advantage over the others. When a substance was introduced, the subpopulations with the most advantageous pumps with respect to that particular substance outgrew the rest.

This is what happened, for example, when they introduced geranyl acetate:

"When the pooled culture was grown in the presence of an inhibitory biofuel such as geranyl acetate, some efflux pumps conferred a distinct advantage. Although all strains started out with equal representation, after 38 h the population composition changed, with cells containing the advantageous pumps becoming an increasingly large proportion of the population. The efflux pumps that enhanced tolerance to geranyl acetate originated from a variety of hosts and include both known and previously uncharacterized pumps."

In their study, Dunlop et al. used a type of membrane transporters called "RND," which are made of big molecules and are only found in Gram-negative bacteria. In a more recent paper [2], Doshi et al. studied a broader set of pumps called ABC, ATP-binding cassette:

"Unlike RND proteins, transporters belonging to the ATP- binding cassette (ABC) protein family are widely found in all five kingdoms of life. They share a conserved structural architecture and specifically import or export a wide variety of molecules and ions across cellular membranes."

Doshi et al. tested whether this family of broadly specific pumps could efficiently mediate the secretion of four different biofuel molecules. Similarly to the Howard et al. paper, they used a bioengineered strain of E. coli and noticed that

"the secretion process was sustained for at least 6 days without the need to replenish the growth medium or culture. Thus, for the same quantity of biofuel produced conventionally, we have a dramatic reduction in biomass scale and significant gain in the ease of recovering the biofuel."

Though my understanding is that work still needs to be done to improve this technique and make it feasible for different types of biofuels, the fact that these transporters are spread across different species makes it potentially translatable to other organisms and therefore of broader use.

On a completely different note, can you guess what the macro picture is? :-)

[1] Dunlop, M., Dossani, Z., Szmidt, H., Chu, H., Lee, T., Keasling, J., Hadi, M., & Mukhopadhyay, A. (2011). Engineering microbial biofuel tolerance and export using efflux pumps Molecular Systems Biology, 7 DOI: 10.1038/msb.2011.21

[2] Doshi, R., Nguyen, T., & Chang, G. (2013). Transporter-mediated biofuel secretion Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1301358110

Fill the tank, please. With bacteria!

I apologize if you've already heard about this, but the paper is really cool and I couldn't resist discussing it here.

Escherichia coli, or E. coli for brevity, is a bacterium normally associated with "bad" things like food poisoning. Even though most strains are actually harmless, even the CDC has a page dedicated to E. coli outbreaks. Since it's part of our gut flora, the lower intestines in particular, it's usually not a good sign when E. coli is found in places like restaurants and cafeterias. (Yuck!)

What's less known to the public is that E. coli is one of the most studied bacteria and makes a great model for mutations, gene duplications, and horizontal gene transfer.

What's even less known is that this amazing bacterium has the potential to save our planet from further drilling. How? By producing fuel. Yes, you read that right: through a combination of gene modifications, researchers from the University of Exeter [1] induced "petroleum-replica hydrocarbons" production in E. coli. These hydrocarbons are structurally and chemically similar to fossil fuels.

In their paper, Howard et al. argue against the current biofuels because they bring additional costs in downstream processing and are not 100% compatible with the engines on the market.

"To overcome the end-user blend wall, it is essential to generate precise chemical replacements to fossil fuels through sustainable means.Retail transport fuels are composed primarily of hydro- carbons (n-alkanes) of various carbon chain lengths (Cn), branched hydrocarbons (iso-alkanes), and unsaturated hydrocarbons (n- alkenes). The ideal biofuels are therefore n-alkanes, iso-alkanes, and n-alkenes that are chemically and structurally identical to the fossil fuels they are designed to replace [1]."

Gasoline, diesel and jet fuels are made primarily of molecules called alkanes, or saturated hydrocarbons. Most people are familiar, or at least have heard of methane, the simplest alkane molecule. These molecules are naturally produced not just by bacteria, but also by plants and insects when they metabolize fatty acids. In 2010 Schirmer et al. described in a Science paper [2] an alkane biosynthesis pathway in cyanobacteria, commonly known as blue-green algae.

"The pathway consists of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes [2]."

Understanding how alkanes are produced and, in particular, which genes are involved in their production, was the first step. The second step was answering the question: can we tweak this pathway to produce alkanes that can replace our current fuels?

Seen under this light, the PNAS study published last March 15 [1] is a bioengineering success story. Howard et al. designed a novel metabolic pathway that forced E. coli to use free fatty acids instead of fatty acid compounds as in cyanobacteria, and produce fuel-like alkanes, what the authors call "industrially relevant, petroleum replica fuel molecules." Once finalized, this type of biofuel will be compatible with current engines and will not need to be blended with other petroleum derived chemicals.

A bit of perspective: though derived from natural and biological sources, biofuels still contribute to pollution, carbon emissions, and global warming. Despite the amicable "bio" prefix, they all come with a non-null carbon footprint, some more than others. The true efficiency of any kind of fuel is the energy they produce minus the energy and costs it takes to derive them. For example, producing biofuels from crops drains precious resources, first and foremost, water, but also arable land, forests when arable land is not available, and food sources in underdeveloped countries.

So here's where biofuels from bacteria have a striking advantage: E. coli is one of the cheapest and easiest bacterium to grow in a lab. It doesn't drain water reservoirs and it doesn't need deforestation to grow. Contrary to most biofuels out there, that have high production and energy costs, the carbon footprint of biofuels derived from bacteria only comes from carbon emissions when you burn them.

And while this is an excellent thing, I still think that the real change we need to make to preserve our planet is to switch to renewable energy.


[1] Howard, T., Middelhaufe, S., Moore, K., Edner, C., Kolak, D., Taylor, G., Parker, D., Lee, R., Smirnoff, N., Aves, S., & Love, J. (2013). Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1215966110

[2] Schirmer, A., Rude, M., Li, X., Popova, E., & del Cardayre, S. (2010). Microbial Biosynthesis of Alkanes Science, 329 (5991), 559-562 DOI: 10.1126/science.1187936

Whole genome harvesting

You think the human genome, with its three billion base pairs and 23 chromosome pairs, is too complex to unravel? Turns out, the wheat genome is six times as big and it's hexaploid, in other words, instead of chromosome pairs it's organized in chromosome sextets!

I've recently discussed genetically modified organisms, crops in particular, and while I still can't provide a definite answer on whether they are absolutely good or absolutely bad, one thing struck me as relevant as I was researching the topic: between climate changes and an exponentially growing population, we are making drastic changes to our planet and resources. While Mother Nature is usually able to buffer changes and constantly adapt to new environments, the changes human kind is bringing upon the planet are happening at such a fast rate that natural adaptation is unable to keep up.

I think at some point we will have to face a hard choice: either starve or give in to GMOs, where by GMOs I mean crops that are bioengineered to yield more in harsher conditions. Again, I'm not saying we should all embrace GMOs as they are healthy and good for us. I really don't know. What I'm saying is that we may not have a choice: in 2009 the FAO estimated that in order to meet the ever-growing demand, wheat production has to increase by 60% by 2050. In the 20th century, the Green Revolution met the increase in demand with the technology known at the time. Today, given the FAO estimate, we may face the need of a second Green Revolution.

With this in mind, you understand the importance of sequencing the wheat genome, a task that is complicated by the complexity of the genome itself. Its three sets of chromosome pairs originated first from the hybridization of two diploid wild grasses, which originated tetraploid wheats (two sets of chromosome pairs) like durum wheat. After thousand years of domestications, these underwent a further hybridization, yielding the hexaploid wheats commonly used today to make bread. Domestication led to a bottleneck in genome variety, nonetheless, the wheat genome has a high percentage of repeats (roughly 80%, mostly retroelements) that yield great variation in length and gene order, making it difficult to sequence.

Despite these obstacles, two papers [1,2] in the latest issue of Nature report using both whole-genome 454 sequencing and shotgun sequencing to assemble the genome of bread wheat and barley. Both sequencing methods have the shortcoming of being applicable to very short regions, and therefore additional work is required to reassemble the full genome out of the various short sequences.

Interestingly, the wheat genome appears to implement a lot of the variation mechanisms I've been extensively discussing here on the blog:

"Several classes of plant DNA transposons and retroelements create and amplify gene fragments, disrupt genes and create pseudogenes, which can influence gene expression through epigenetic mechanisms [1]."

Similarly, in barley:

"Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation [2]."

Brenchley et al. [1] conclude:

"Major efforts are underway to improve wheat productivity by increasing genetic diversity in breeding materials and through genetic analysis of traits43. The genomic resources that we have developed promise to accelerate progress by facilitating the identification of useful variation in genes of wheat landraces and progenitor species, and by providing genomic landmarks to guide progeny selection. Analysis of complex polygenic traits such as yield and nutrient use efficiency will also be accelerated, contributing to sustainable increases in wheat crop production [1]."

[1] Brenchley, R., Spannagl, M., Pfeifer, M., Barker, G., D’Amore, R., Allen, A., McKenzie, N., Kramer, M., Kerhornou, A., Bolser, D., Kay, S., Waite, D., Trick, M., Bancroft, I., Gu, Y., Huo, N., Luo, M., Sehgal, S., et al. (2012). Analysis of the bread wheat genome using whole-genome shotgun sequencing Nature, 491 (7426), 705-710 DOI: 10.1038/nature11650

[2] Mayer, K., Waugh, R., Langridge, P., Close, T., Wise, R., Graner, A., Matsumoto, T., Sato, K., Schulman, A., et al. (2012). A physical, genetic and functional sequence assembly of the barley genome Nature DOI: 10.1038/nature11543

GMOs love me, GMOs love me not..

I've been asked to discuss genetically modified foods and I confess I've been procrastinating. Why? Because I don't have an answer on whether or not GMOs are good or bad, and I can't offer one. But, what I can do is offer a few thoughts. Food for thought is usually super-natural, organic, and pesticide-free, so here it goes. :-)

1. What are GMOs?
Technically, all domesticated plants and animals are "genetically modified" since, rather than letting the species evolve through natural selection, mankind has steadily selected offsprings according to some man-made criteria. However, today's technology allows us to artificially modify an organism's genome. The difference between the two is not just in time scale: when selecting crops, or, more in general, any organism, generation after generation based on phenotype, uncharacterized genes are introduced in the species. Genetically engineering, or bioengineering, however, introduces a few well-characterized genes (often from a different species) into the organism. In a way, this is no news: gene therapy creates genetically modified organisms. Humanized mice are created in labs to test drugs and other therapies. The question of whether or not GMOs are good arises in the food industry. Are they safe to eat?

2. The Cartagena Protocol on Biosafety
As California gets ready to cast its vote on Proposition 37 [1] (which would require foods to denote their GMO content on the labels), it is good to review what currently is in act to "protect" us from possible hazards. From Wikipedia:

"The Cartagena Protocol on Biosafety is an international agreement on biosafety, as a supplement to the Convention on Biological Diversity. The Biosafety Protocol seeks to protect biological diversity from the potential risks posed by genetically modified organisms resulting from modern biotechnology."

"The Biosafety Protocol makes clear that products from new technologies must be based on the precautionary principle and allow developing nations to balance public health against economic benefits. It will for example let countries ban imports of a genetically modified organism if they feel there is not enough scientific evidence that the product is safe and requires exporters to label shipments containing genetically altered commodities such as corn or cotton."

3. Why are foods genetically modified?
For a number of reasons, some good and some not so good. Some are just practical reasons in a world that, whether we like it or not, is getting more and more "globalized": the first bioengineered produce was a tomato designed to have a prolonged shelf life. Some crops are genetically modified to resist harsher herbicides and pesticides. Others, are genetically modified to desist bugs from eating them. For example, genes producing Bt toxins have been introduced in cotton and corn. These toxins kill caterpillars that would otherwise eat up the whole crop. Notably, the modification benefits not only the genetically modified crops, but, since it reduces the global population of harmful caterpillars, it also benefits the non-modified crops.

I expect foods that can resist herbicides to be soaked in chemicals. On the other hand, if a crop is genetically modified so its flowers/fruits/seeds no longer offer a viable environment to certain parasites, I expect those foods to be pesticide-free. Yes, I'll take a few modified genes over harmful chemicals. Bottom line: NOTING WHETHER OR NOT A CERTAIN FOOD CONTAINS GMOs DOES NOT HELP. What you should really demand in a label is WHY SUCH FOOD WAS MODIFIED AND WHAT WAS ACHIEVED THROUGH THE BIOENGINEERING. Notice that while the Food and Drug administration currently does not impose any GMO labeling, their guideline recommendations state that the GMO content be noted, as well as the reason why the food was modified, and what was achieved through the modification.

4. Genetic homogeneity is bad
Rice is one of the most consumed crops in the world. Again, from Wikipedia:

"As of 2009 world food consumption of rice was 531,639 thousands metric tons of paddy equivalent (354,603 of milled equivalent), while the far largest consumers were China consuming 156,312 thousands metric tons of paddy equivalent (29.4% of the world consumption) and India consuming 123,508 thousands metric tons of paddy equivalent (23.3% of the world consumption). Between 1961 and 2002, per capita consumption of rice increased by 40%."

Rice is also highly "domesticated", as it has been selected over thousands of years to fit human needs. Currently, there are 20 different kinds of rice, but, according to FAO, the Food and Agriculture Organization, "It is estimated that not even 15 percent of the potential diversity has been utilized." This is a THREAT to food security. If a pesticide-resistant parasite were to attack rice crops, it'd be lethal to the vast majority of rice varieties currently harvested. Heavy use of pesticides favors the selection of pesticide-resistant organisms, while domestication favors genetic homogeneity in crops. This is NOT a good combination. Another reason why, between GMOs and pesticides, I'd favor GMOs. And if GMO research can prevent a pesticide-resistant organism to wipe out 50% of the world-wide food, hey, who's to complain?

5. Knowledge is NOT power if that knowledge is poorly understood
We live in a strange era when technology leaps forward at a higher speed than our ability to comprehend its output, especially in the field of genetics. We have loads of data we don't quite know how to store, let alone analyze. It's getting cheaper and cheaper to have a full human genome typed and companies are advocating that we do it for every individual. But are we capable of understanding the data? Last week I posted a shocking story of a boy discriminated because he carries a recessive mutation for a disease he doesn't have and he's at no risk of contracting (that's what recessive means). The Internet is full of bogus info on genes, genetics, mutations, etc. There's more noise than ever, giving people the illusion that they know when in fact they don't. I fear that the same will happen for GMOs. Once those labels come out, will people be able to understand what they mean? If Prop 37 will only require a "content" statement without a "reason", for example, will the information be really useful or will it just generate a stigma?

You now see why I cannot tell you whether GMOs are good or bad. They can be both! (Aren't we all?)

Food always has a higher impact than other things, but if you think about it, there are so many things that we've introduced in our daily lives in the past few decades that we simply don't know whether or not they are good IN THE LONG RUN: wi-fi, for example. Cell phones. Chemicals in skin products, from sun protection to cosmetics. I'm afraid the next generation will be the test. So the real question is: do we want to experiment with our children as guinea pigs? Sadly, when you put it in these terms, it seems to me it's too late to go back. The experiment has already begun.

If these few thoughts weren't depressing enough, read Pamela Ronald's review, referenced below [2]. One of the great points Ronald makes is that we are changing our climate and environment much faster than ever before (thanks to climate change and an exponentially growing population). Natural selection can't keep up with the pace, hence

"an important goal for genetic improvement of agricultural crops is to adapt our existing food crops to increasing temperatures, decreased water availability in some places and flooding in others, rising salinity, and changing pathogen and insect threats."

The review is clearly biased in favor of GMOs and it lists several benefits from such procedures. While advocating for adequate testing on every newly modified organisms, it also reports that all genetically modified crops tested so far have been deemed safe and substantially no different than conventionally selected crops "in terms of unintended consequences to human health and the environment."

Bottom line: I can't tell you what to vote on Prop 37 and I can't tell you whether or not you should avoid GMOs. Just read as much as you can and be sure to form your own opinion.

REFERENCES:

[1] Baker, M. (2012). Companies set to fight food-label plan Nature, 488 (7412), 443-443 DOI: 10.1038/488443a

[2] Ronald, P. (2011). Plant Genetics, Sustainable Agriculture and Global Food Security Genetics, 188 (1), 11-20 DOI: 10.1534/genetics.111.128553