Synthetic Biology: Principles and Applications – Jan Roelof van der Meer

Hello, my name is Jan Roelof van der Meer, I'm a professor in microbiology at the University of Lausanne in Switzerland. Today I would like to talk to you about synthetic biology. About the principles of synthetic biology, and some of the applications. Some of you may have very different perspectives and ideas about what is synthetic biology. You may have heard of the word, you may have associated it with plastic organisms or with organisms doing various strange characteristics. But probably, this is not what synthetic biology really is. So my goal of today is to explain to you the concepts of synthetic biology and contrasting them to the normal way that biologists work when they try to understand living beings. After that, I will tell you something about research directions that are ongoing in synthetic biology, and I would like to explain some of our own work, which is about synthetic bioreporter cells that we think are useful for environmental purposes. So, if we think about biology, it's really about understanding living organisms in all their aspects. So you may think that biology is about going out into the jungle and looking at elephants, but as a microbiologist, we often look just at bacteria, microscopic organisms.

So what you see here is this small growth chamber that we developed in order to look at the behavior of single cell bacteria that you can see here as these small rods. And what this small instrument is doing is that we can feed the bacteria from the left side and then look at their behavior on the right side. So it's really very simple in the sense of looking at what the organisms are doing. Biology often uses observation, just observational techniques to study behavior. So here on the left side, you can see for example how bacteria, even though they're extremely small, use a flagella to move themselves forward in search of nutrients or conditions that they have. You can see the spirally movement of the flagella that propels the cells in one direction, and if they want to change the direction, you can see that the flagella become disordered and they can rotate the cell to go in another direction.

You can see on the other image here more closer, that this is an observation of a single Daphnia individual. And Daphnia is a small water creature that lives in most freshwater habitats. And what you see here is the movements of its legs and of the heart and of the internal organs. So the organism is sufficiently transparent that you can keep it under the microscope. Here we keep it in a small cage, where the organism sits and is fed with fresh water. And we can observe how it reacts. So observation really is one of the critical tools that biologists can use. Another tool that biologists use a lot is understanding from the creation of mutations. So what are mutations? Mutations are changes that we make in the DNA, the hereditary material of organisms. Again here you see a very simple example, on the left side you see the cell of a wild bacterium called Bacillus subtilis. Which is a bacterium that normally dwells in the soil, it can make spores, it knows how to survive very well. In order to understand how this cell divides, researchers have made mutants that cannot make proper cell walls.

So for example, if you look at this particular cell here, it's completely round and blown up because it carries a mutation in a gene that is essential to make the cell and that otherwise maintains the cell as a nice rod shaped structure that you see on the left. So by knowing where these mutations are, we can try to understand how the organism organizes itself and makes this cell wall. The third important aspect that biology uses is what we call dissection. So we like to take things apart in biology in order to understand. This can be an anatomic dissection, like you see here for a bee where investigators of our own department dissect the bee to understand how the bee gut and the internal organs of the bee work. And how they interact with bacteria that live in the gut of the bee. So you can see here, a researcher preparing the gut of the bee in order to understand this. It's not only anatomic dissection that biologists use, but more and more, we also use genetic dissection. So we like to understand what the DNA is made of in every living organism and how this contributes to the whole body plan and how the whole functioning of that particular organism.

Maybe you have seen genetic dissection of DNA before, what you can see here for example, is a culture of cells on the left, it looks like if you have a soup, a turbid soup. This is because the soup culture contains millions and millions of bacterial cells that you can break open by lysis and then you can isolate the DNA that in solution looks sort of like this fluffy solution. This fluffy material, this white-ish material. If you put this white-ish material under a microscope, here under the atomic force microscope, you can see that it forms sort of a chain of pearls that you can observe. And you can draw certain conclusions from it, but more importantly, for DNA, we often look at the gene sequence.

So we take the DNA apart, we determine base by base what the DNA looks like. And that's shown here in the trace below, where every peak that you see in a different color, in red or green or blue, means a different base of which the DNA is made up. Now if we take all that sequence together, so then we try to convert it to code, just the code of A, C, G, and T, you can get a very nice and thick book. This is the start, if you like, of a genome sequence of a single bacterium. This bacterium doesn't have a very big genome, it's only like 6 million characters. But if you think about this page containing 2000 characters per page, then you would still need 3000 pages to print that whole bacterial genome, which is quite a thick book. And if you think that the human genome is a thousand times bigger, then that would be a very big genome.

So normally we don't print that out, because it would take too much space. Now the real goal in biology, in particular molecular biology is to understand what does this sequence actually mean. All these letters that are there. What do they do? How can this be the important plan for the bacterium or the living organism that is there? So what we often do is we try to gaze into the sequence and do an analysis of important features that this sequence can contain.

So as you know, the sequences contain for proteins, for RNAs, there's signals on the DNA that are important to direct certain proteins to actually read the instructions in the DNA and form the parts for the cell that are needed. So what is really important is that we understand what such a DNA sequence means. And as I said, this could mean a really big sequence. So when we look at this particular part, you can see some of the things that biologists try to interpret. This is the case of a bacterial genome. So what we are looking at here in what is called reading frame, is actually the region that is needed for the cell to recognize, oh this is the part of the DNA where I have to make an mRNA and then a protein. A reading frame has to have a start, like here is shown at the ATG, that's the start of that reading frame. It's a signal to start building the protein where it's needed. But then there are also other parts that are needed, for example, what is shown here as an RBS. This is a site that is recognized by the ribosomes, the factories that produce the proteins, to begin the synthesis of a protein.

And then there are often other parts on a sequence that do not code directly for a protein, but are important for other proteins to know where to start doing the task they have to do. So for example, here in green is the protein binding site, it's a transcription factor binding site that directs the machinery toward expressing that gene. Next to it is a promoter sequence, that is a signal for the RNA polymerase to start transcribing that gene and so on and so forth. Now this is really the basis, or this is really where biology ends and where synthetic biology starts. Because synthetic biologists start to interpret this sequence in a different schematic way. So one of the concepts of synthetic biology is really that you break the DNA down into biological parts. This can be DNA parts that you can assemble in a particular way, or it can be protein parts if you want to profit from these protein parts. So if we look again at this sequence that I just showed you in a different way, in a very schematic way, then it may look for a synthetic biologist like this.

A gene, so a coding region that is needed for a protein, will look like a small arrow here in green or there in brown. That codes for protein 1 or protein 2, depending on what we need. The synthesis of those genes are driven by promoters that we display by different other arrows, here in small black arrows, and we have important signals for the ribosomes to start the translation of such proteins that are listed here as RBS. And there may be other things that a synthetic biologist needs like here, a binding site for a regulatory protein, and here a terminator that's a signal for the RNA polymerase to stop. So it's really important to try and understand. We can decompose the sequence into parts that we can study as they are in a living organism in the particular way that they appear, but we can also move them into different parts.

So if we take this sequence apart, then we see really what the circuit parts are so that the synthetic biologist would need. So we may need a part for genes, we need a part for ribosome binding sites, promoters that are signals, terminators that are signals, binding sites for transcription factors on the DNA, these are the parts that we need in order to assemble something. The protein part that we need would be a structural protein, a regulator protein that we can see that is important to signal the cells "yes now you start transcribing that gene or not." We need transcription factors, we need sensory proteins depending on what we actually want.

So it's really important to realize that we can go from the sequence to the parts, we can study the parts and then we can put them back together in a different way. Now the second concept that is very important for synthetic biology is rules and models. So we do not only like to dissect the sequence and know the exact sequence of the A, C, G, and T's in the genome of an organism or a part of DNA that we want to construct, but we want to understand how does this sequence work together. So which are the rules that the cell is following in order to make this sequence functional? So for that, synthetic biology uses certain rules. This could be logic rules like that gene is on or that gene is off. It could also be models like shown here in the back, that tries to predict how a particular stretch of DNA and promoters and terminators and binding sites is working for the cell.

Now if we go back to that same DNA circuit, the same stretch of DNA that we have seen before, with the two genes in green and in brown. And the different parts that are needed to operate this particular gene circuit, then it means for the cell the following, you can see that in steps 1, 2, and 3. The first signal for the cell so that it starts to interpret this DNA sequence is that it will try to transcribe this particular gene. It does that because there is an RNA polymerase coming. The RNA polymerase starts at the promoter and then transcribes that gene until it reaches the terminator.

This mRNA is then translated into a protein that you can see here schematically in green. What this protein is doing is that this protein will bind to the DNA at the particular site that is here in green. Now that protein's not just any protein, it is a sensory protein with also activating functions, so it is capable of sensing for example, a particular chemical that interacts with this protein and then tries to attract RNA polymerase again, but to a different promoter. So what this protein is now doing is that it attracts RNA polymerase, but to a promoter that is here. And then when RNA polymerase is there, it will then transcribe that gene and make that particular protein. So this small schematic structure is actually giving some instructions to the cell, start here automatically, make a protein, bind that protein that can intercept that signal, and then transcribe another protein.

So a very simple thing that follows a certain set of rules. You can put these rules in a kind of model if you like. If you have these simple circuits, you can sort of by modeling, try to predict what they're going to do. Here's an example of two simple circuits, in one case we have the two genes that are located in the opposite direction.

In the other case, we have the same genes but located next to each other. Now the rules that this small circuit says is that this particular gene codes for a protein that will then inhibit the transcription of the other gene here. So in one case, this protein will inhibit its own synthesis and the gene the gene that is in yellow behind it, in the other case it, it cannot inhibit its own synthesis because it is not binding there, it's not influencing this particular promoter that would transcribe itself. Now the model now would predict that in the case where you have this feedback, where FB means feedback loop, then this would be dependent on a signaling molecule, that is in this case arsenic.

And as a function of the arsenic concentration that is shown here below, you can see that the more arsenic you add to the system, the more of this protein ArsR you get. And the more of this protein GFP that you get. In the case of the uncoupled systems, so UN means uncoupled here, then this gene is not under its own control, but it's under the control of something else. You can see that it's always produced at a constant level, which is independent of the concentration of in this case, this arsenic or AsIII. But the other protein is still under the control of this AsIII, so as you can see here, this increasing amount when the concentration becomes higher. So this is a simple model, it's a very simple genetic circuit as we call it. It gives a set of instructions to the cell and the cell will carry out these instructions if it is properly equipped. The third concept of synthetic biology is really standards. Standards? That sounds very, very weird.

Why would you need standards in biology? Well, think about it. Synthetic biology has a fair amount of relation to electrical engineering, where people were working in the beginning with electricity and trying to harness electricity in forms that are useful. Like cameras, like televisions, and so on. So the industry and the people had to adopt certain standards that we now know as electrical plugs. Now the electrical plug may still be different between Europe and the U.S., but the essence is that there is an electrical plug you can plug something in there and it gains the electricity and can work. In synthetic biology there is a similar concept in order to try to make it possible that people from different laboratories and different industries can work together on the same parts.

So maybe we are thinking about standards for gene expression. But how would that look like? It's not electricity, it must be some biological equivalent of electricity. And the plugs? What could they be? They could be small fragments like here, promoter sequences that can be adopted into one system or another system. So standards is really an important part for synthetic biology. Now having explained all this, what is synthetic biology really about? So what is synthetic biology hoping to achieve? There's two main things really, at this point. One is that we can understand complex biological processes not by dissecting them as normal biologists do, but by reconstructing them. So we take parts and we build something that is more complex, like here schematically shown for Legos. It looks very much like Legos. So understanding biological processes not by dissection but by their reconstruction. The second thing that has appeared in synthetic biology and that is maybe not so different as people may know from genetic engineering or so is to facilitate the construction of complex biological processes that carry new functionalities. Not just producing one protein but producing a complex pathway that you engineer into the cell that was not previously possible.

So these two things are really what synthetic biology is nowadays trying to accomplish. The engineering idea, as I said, is really rather similar to what electrical engineers do. They have their parts, they can be small transistors, transformers, capacitors that they put together on an electrical board. These electrical boards, if you put them into your computer, can give your computer certain instructions. Biologists and synthetic biologists are trying to do the same. Take biological parts with some rules, models, and engineering, we put them together. And then we try to verify what this construction really is doing and what it means. Now current research activities in synthetic biology go consequently in all directions, I would say. There are groups that work on making standardized parts, making new models, trying to come up with complex engineering strategies to put these parts together. That is really important, because if we want to play with parts, we actually need to have parts. So the more parts we have, the better they are characterized, the better we can produce new structures in synthetic biology.

The second part of synthetic biology has really started off with DNA synthesis. So previously in genetic engineering, it was really difficult to make mutations and really cumbersome and took a lot of time. Now there are DNA synthesis companies and biologists will simply write down their sequence, send it by their computer to the DNA synthesis company who will actually make the construct, and that facilitates largely to put parts together in a particular way. So consequently, there are people who try to design whole genomes, which is still an important and challenging task. Because we do not understand all the rules very well to actually be able to put genomes together. In some cases, people also use genome parts like complex phenomenon that the cell does. If you remember the example of the swimming cell, so the flagella synthesis even for a bacterium takes a lot of power, it's a very complex process with many proteins.

So that's something that a synthetic biologist may try to reconstruct. The third thing is something that looks really bizarre if you think about it. It's the production of minimal cells and host production platforms, so synthetic biologists have adopted this terminology that's called "Chassis," almost like a car factory. You have your chassis that you can put in this kind of chair or that kind of chair, and it doesn't really matter because the car is still running. So the same idea appears for biology as well. You can make bacteria or yeast that are just a chassis needed to make the motor for the cell. And everything else you can plug in, colors, pathways, things and so on. So for that, very often people find that the living beings that exist naturally are way too complex. They contain viruses, they contain things that you wouldn't really need, and that is why they want to design minimal cells that have been devoid of all the parts that are not really needed.

A fourth direction in synthetic biology really tries to go even beyond it, that is trying to make protocells and artificial life. There is a huge interest in trying to understand where is life coming from. We do not know, but synthetic biologists may be able to recreate certain life forms and that would help enormously to try and understand where is life coming from and what are the different paths that can lead to life. Finally, there's a lot of effort in what's called Xeno-DNA, and this may be sort of your fantasy dream strains of DNA. But what it's really about is that biologists and synthetic biologists are saying, you can alter DNA, you can alter proteins, in that you incorporate different types of amino acids that the cell normally doesn't like, but it could be really important to try to incorporate all these into proteins because it could give new functionalities to proteins that we cannot currently make.

So this is the xeno-DNA/biology. And finally, there's an important point that comes with synthetic biology that allows biology to attach to a do-it-yourself community. So many people also amateurs become interested in biology because of the efforts in synthetic biology. Trying to understand biology, making simple instruments that you can use in organized groups and so on, to try and understand biological phenomenon. So this is really an overview of the general research activities in synthetic biology. I would like to pick just one particular application. To give you some idea of things that people are dreaming of, and this is obviously one of the things where you may say, okay will these dreams finally come true? But this is a bit of marketing, if you like, by the biologists and the engineers that are behind it. So there's a lot of hope that synthetic biology will be able to help producing new things that will be useful for human health, animal health, there's obviously a lot of money going into it. In terms of pharmaceuticals, vaccines, maybe gene therapy, tissue engineering, probiotics, diagnostics, and so on.

Another area of importance is agriculture. Try to improve plants that are resistant to diseases, resistant to drought, that give better feedstocks for animals, that can maybe help sequestering CO2, chemical production, diagnostics. Then there are things in industry, you may have heard about bioenergy and biofuels. Things that can become very important if synthetic biology is able to create better organisms that do these kind of conversions with higher efficiency. Production of bulk chemicals is very important because maybe at some point, we'll run out of oil and we need alternatives to actually produce the chemicals that we need daily.

Specialty chemicals, new materials, people are thinking about building DNA and proteins together to get new kinds of materials that might have properties that we have not seen before. And there's also applications in the environment, like biosensors, bioremediation, waste treatment that may be helped by engineering specific organisms that do tricks that we cannot normally achieve in the natural conditions. So let me explain to you just about one of the things that we do in our own lab, which is called bioreporters. These are really very, very simply engineered bacteria cells.

Bacterial cells that are not pathogenic, harmless in the lab. And what we can do is that we can equip them with different colors like here, this is called bioluminescence, it's really a cell that gives off light. Or with fluorescent colors, you shine light on them and they produce another color back that you can measure. Or just regular colors like blue, red, green, and so on. The idea with these bioreporters, as we call them, is that the cell can signal for us the presence of, for example, a toxic chemical in the environment. And then what the cell is doing is it has a small circuit inside, so it will recognize the compound that will diffuse inside the cell and then this compound is bound again by one of these sensory proteins that I talked to you about before that can bind the DNA and can direct the synthesis of a new protein in the cell.

And the new protein is often one of these proteins that we have seen here, that gives off light or fluorescence and so forth. So we think that these are very simple cells that can do very useful tricks for us, because they can help us to make analytical devices to sort of interrogate parts of the environment where we think there is contamination that may occur. One of the systems that we have been working on is to construct cells that would detect arsenic. So you know arsenic from the novels of Agatha Christie, it's a really nasty toxic chemical. But unfortunately, it has not only been used in novels of Agatha Christie, but large areas in the world are contaminated with arsenic from natural resources.

So it's an abundant metal that exists in the earth's crust and can come up in the ground water. And people like here, shown in this picture in a village in Bangladesh, suffer enormously because they do not know if the drinking water that they take from their household pumps is actually contaminated with arsenic or not. So we sat together in the lab and with a small spin off company called ARSOLUX, that is a collaboration of the Helmholtz Institute in Leipzig in Germany, to make bacterial systems that would be able to measure arsenic in drinking water. And then could be used on the field to measure the water that comes from the pumps and analyze this for arsenic.

So what we do is we make small glass vials, and you can see here sort of a powdery stuff. This powdery stuff is really the bacteria that are dried inside such a vial. The vial is closed with a stopper, and that's important because that makes it a closed system and the bacteria cannot escape. We inject the water directly through the stopper inside it, this reconstitutes the bacteria, as you can see here.

It makes this sort of watery suspension, if there is arsenic in this water, the bacteria will react to it and will start to glow. So they will make this famous bioluminescent signal that you cannot see by eye unless you are in a very dark chamber. But you can very easily do this by putting these small vials into a small instrument that's shown here, that is called a luminometer. This is a portable luminometer that we can use in the field.

It has a battery capacity, you close the cap, you wait a little while, and it measures the light that comes from the cells. So what we have been able to do is, if we are in such villages, then we can sample all the wells from those different households. And that is really the problem, that they don't have a central drinking water supply, but individual households are pumping and you have to test all that water. And not just once, but multiple times. So what we can do is go into such a village, fill all the different vials that are necessary for each of the pumps. Fill them one by one, and then wait until the cells react, and then measure them one by one by one. And in an afternoon, you can measure all the water wells in the whole village. Obviously if you try to do such a test, it's very important that you can actually show that this is working.

So in the first test that we tried to do, this was done in Bangladesh and in Vietnam, in different settings with different types of ground water. We compared that at the same time, the response from our engineered bacterial cells with the response of classical chemical analytics by ICP-MS, or with atomic absorption spectrometry. And as you can see here, there's a very good dependence between the signal that's given by the biosensors and the signal that is given by the chemistry.

So there is almost a one to one ratio of the concentration that you measure by chemistry and with the biology. And that tells us that this method is potentially very good and very interesting because the bacteria multiply by themselves, so to produce such a biosensor is extremely cheap and doesn't require a lot of engineering. Whereas to make a GC, MS or atomic absorption spectrometer, it costs a lot of money and you cannot deploy it in the field. So that is why we think that this test could be very interesting to do this. As another example, we use bioreporters to measure pollution at sea. So here we engineered a set of bacterial reporters that could measure different compounds that come off of oil, like alkanes, solvents, basically aromatic hydrocarbons. For this we worked together with the Dutch government on an exercise in the North Sea. The Dutch Government has what is called responder vessels, they go out whenever there is an oil pollution and they scoop the oil and bring it back to the refinery if they can.

But much of the oil, particularly smaller spills, go undetected and floats there. And nobody really knows how dangerous this can be. So what we set out to do with these responder vessels is that we got permission to actually make an artificial spill out in sea with a limited amount of crude oil. And then we went onboard with our small portable luminometer that you see here again, the different cell lines in the vials that we can directly incubate with the sea water to try and measure what is the oil pollution that really occurs at the sea.

This sampling was quite challenging, as you can imagine, we had to go out with a rubber boat from the responder vessel to actually approach the oil slick, that you can see for example here. Because the ship itself is so big that it cannot go into the oil slick, because otherwise it would be horribly contaminated as well. So here is an example of the results that we found in these exercises. Again, in the top you see the chemical analysis, and in the bottom, you see the analysis of what we call the reporter cells that were done onboard.

The chemical analysis was obviously extremely good, but it took two months to actually get to that. Whereas the bioreporter signals could be obtained directly onboard the same afternoon. So here is shown the results of two experimental spills, we had one opportunity in 2008. And one opportunity in 2009. And then there are the spills that we encountered on the way, because the North Sea is a very busy traffic route. And ships from time to time, they clean their insides and they throw away overboard some oil, which we can also analyze. So importantly, what you can see here in the diagram below with the different colors is the different parameters that we measured with the reporter cells. So you can see that in all cases, our samples from the sea water that were far below the oil slick that we measured important concentrations of toluene, benzene, methylene, alkanes, etc … So that told us again that what we measured with these cell lines is very, very relevant and can help to address the situation of samples at the site immediately.

And we hope that these sort of results are convincing to the authorities to give permission and perhaps to companies, to say, oh this is an important way of trying to analyze and apply synthetic biology efforts. So finally, I would like to give you sort of a prediction or report. So this is a report that was commissioned by the European community to estimate the global value of the market for synthetic biology. This report was done in 2011, and obviously these things are always a bit predictive in the sense that maybe they're not too conservative, but you can see that the estimates for 2011 were already $1.6 billion USD in various fields like pharma, chemical products, agriculture, and energy.

In 2016, it's rising up to $10 billion. So this is really something that everybody has high hopes, that synthetic biology is going to be a globally important market. I hope that I have shown you a little bit about how synthetic biology works, how the concepts work, with the bottom up construction, not the dissection and destruction of organisms, but taking parts and building something again. Synthetic biology has many useful applications, potentially useful and that's how the research is going. Some of the things may not make it in the end, whereas other things come surprisingly and will in the end deliver important results. Several results are within close reach, so it's not something that we have to wait 25 years to deliver. No, no, there's important applications and some of them, like we demonstrated with the small bioreporter cells to measure the environmental quality can be used immediately. Thank you very much for your attention..

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