The Essential Unknowns/Set Theories

Thoughts and framing of how to figure out what the essential genes are.

Build-A-Cell Team

The challenge of discovering the function of the essential unknown genes is tightly correlated with the project of building a functioning synthetic cells. This is a collaborative and multidisciplinary effort of the multiple research groups across the globe, as achieving of the objective of building an artificial cell is far above capacity of an individul, or a single research group.

During the Build-a-Cell workshop held at University of Minnesota on August 6 2018, we asked some of the leaders of the Build-a-Cell project for their thoughts about the building the synthetic cells, role of investigation of the essential unknown genes, and the biggest obstacles on the way to build a cells. Their diverse perspectives should inspire the reader to appreciate the complexity of the challanges related to building a cell, and to look deeper into the problematics.

Drew Endy, Stanford University

(Coming soon)

John I. Glass, J Craig Venter Institute

Highlights:
- Mycoplasmas are the most suitable organisms for studying of the minimal genome because of their small genome size and simplicity.
- Revealing of the function of the essential unknown genes can have paramount impacts, ranging from new antibiotic targets to the description of the new disease states.
- With all the tools we have in modern biology today, we should assimilate the multiple streams of knowledge and information about these genes to discover their function.

What is the best way to build and understand the cell?
The idea has long been the building of a minimal cell. In the 19th and 20th century, the physicists and chemists understood the nature of matter by studying the Hydrogen atom, the simplest of all possible systems, reasoning that what is true for Hydrogen would be true for more complicated atoms. But while the mathematical description is possible for Hydrogen and for many of the things that have been done, it is still not doable for Helium, Carbon, or Uranium. So think about trying to start with simple biological systems, which is why for the last 80 years, the biologists have been thinking about this idea of building a minimal bacterial cell with the absolute simplest metabolism, the simplest machinery possible, so that you understand the systems with minimal redundancy in order to see how things interrelate. And that’s what led me to think about doing a career working with Mycoplasmas – they are the simplest organisms that we know of in nature at this point. Because of the incredibly stable niches in which they have evolved to live, they have been able to throw away the most of their genomes and the mentioned simplification takes place. What we tried to do to build these synthetic organisms is that we further streamlined their genomes. So rather than taking an organism designed to live in a goat or evolved to live in a goat, we have gotten rid of the genes necessary to avoid the immune system and we had an organism that really can only live in one environment, which is the laboratory media that we provide. And so the simplification, I think, is the key towards building cells. And then you’ve got to think about using those to understand the first principles of the cellular life.

How would you discover the function of the essential unknown genes? Is it even important?
The organism that we built had 473 genes. We were astonished to find that out of 473 genes, 149 we did not know the function of - meaning that the of the third of the genes of this organism, and this organism has a very low percentage of genes of unknown functions relative to other organisms. You may think that it is a high percentage, but if you set the criteria very high, saying that you have to clearly understand what it is doing in order to classify it as ‘known’, for a great number of the genes of any organism we do not know what they do. And that means that a huge amount of the biology of these organisms is a mystery to us, in terms of what these proteins do, and how they function. The importance of this is hard to estimate; if you think about bacteria, there could be the new antibiotic targets. If you think of medicine, then if you are oblivious to many of the biological processes going on in cells, there could be disease states, there could be control issues that are important from Mycoplasma to men that you really do not know about. And so how do we figure out what these genes do? The traditional way to figure out what these genes do has been one gene - one graduate student - one PhD to get to this. That still works, but with all the tools we have in modern biology today, it is practical to think about assimilating multiple streams of knowledge or information that we are accruing about these gens in order to do a better job of providing clues to figure out how you might characterize genes. And I think that it is going to be the tendency at least to get it down to a very small number of genes. We started two years ago with 149 genes of unknown function. Now we have a pretty good idea of what 45 of these genes do, so we have been able to drop this amount by about a third – not quite a lot, but the process is moving along.

What is the biggest challenge on our way to build a cell?
I wish I knew! There are so many challenges – imagine that from membrane materials of the shelf, you can make membranes that are comprised of appropriate lipid chemicals that you need. And you that you build these vesicles, and then put in a transcription-translation (TX-TL) system that will allow you to put proteins into those membranes that allow the exchange of metabolites necessary for life across them. So you have got a cell-free system with transcription-translation, and you build ways to get import and export of the necessary metabolites to keep the system functioning for some time, and then you manage to find the way to install a genome capable of programming a living cell into this. What if you get all of that done and it still does not form a cell? What if there is more to it in the sense of establishing the dis-equilibrium across the membrane surfaces that are characteristic of life? I worry that the hardest part of this is something that we are just not considering yet. But in theory, also, it seems like if you put all these pieces together, you might have this cell that would live and from there we can think of building more complicated organisms.

Kate Adamala, University of Minnesota

Highlights:
- The best way of understanding cells is to build the entities that resemble natural cells from scratch.
- Expressing the essential minimal genes in the artificial liposomes can be the best approach to reveal the function of essential unknown genes.
- The biggest challenge on our way to build a synthetic cell might be the self-replication.

What is the best way to build and understand the cell?
I do not know – that is a kind of purpose of the Build-a-Cell community meetings - to figure out what would be the best way to doing it. To me, the best way of understanding cells is to build from scratch entities that resemble natural cells - either in function, in part of function, or in a structure. Once we have them, we can engineer them, we can reverse-engineer them and we can take them apart and see how they work. So that would be the best way of understanding the natural cells and in the process of that you would end up building an artificial cell. I do not know if that is the best way, but that is the way I would go about it.

How would you discover the function of the essential unknown genes? Is it even important?
It is extremely important. Discovering functions of each of the essential gene is absolutely crucial for building the cell from scratch. And I think that best way to approach it is to reconstitute essential minimal genome in a completely artificial synthetic cell system. Basically express those genes in artificial liposome and see what functions we are missing as we are removing one or the other gene cluster. It is hard and lot of the time you do not know what is the function you are looking for, but this is the approach that I think would be the fastest, since we already done all the simpler things like gene mining and trying to align those genes with other families, so now we just have to do it experimentally and I think that this is one of the ways that may actually work.

What is the biggest challenge on our way to build a cell?
I think self-replication. To make the biological system that is capable of self-replication, requires making ribosomes that can make more ribosomes and making metabolic systems that can make more lipids, that can make more nutrients and that can break down and remove the waste products. And all of that is necessary for the ability to spontaneously grow and reproduce the synthetic cell. You also need to have a cell cycle that provides a clock that initiates the division. So all of those elements have to come together in order for the self-replication to happen so that’s why I think it is the biggest challenge because it kind of unifies all the other challenges we have been talking about. And once we have a self-replicating cell, most of people would argue that it is alive, so that is the biggest, but also the ultimate challenge.

Laurie Zoloth, University of Chicago Divinity School

Highlights:
- The ethical challenges of building a synthetic cells are profound and have to be thoroughly discussed.
- Ability to create a new living organism would give humans the power that is unprecedented and the appropriate control framework will need to be developed to manage this power.
- The process of building the cell will require the collaboration among scientists, theologians, and philosophers.

What is the best way to build and understand the cell?
There are two ways of thinking about it. The traditional way has been to look at the existing cells, see how they act in the nature and what their properties are, measure their different activities and functions, and to understand from top-down, so to speak, what you have here, how it works. And that is mostly what has been studied through the 17th through 20th century – you just observed and measured, and maybe you poked it a little bit to see what would happen, to have the empirical reaction. The synthetic biology really wants to start from the bottom-up, thinking about disaggregation of the function and the properties of the cell and to see what emerges if you put those things back together. So it is working with that sense of emergent property and the sense of building it from pieces rather than looking at the existing functioning cell. So that is interesting – that is an interesting way to think about the knowledge; it is very Greek. Greeks really believed in Techne, they wanted to know by learning, by the craft of making, not just by thinking about theories, but about how the practice works. And so this is very “Techne” kind of project.

How would you discover the function of the essential unknown genes? Is it even important?
It is important, because more of the world is unknown than known. And that’s been something that has been true since antiquity. We know much more about how the world works, we know about the existence of atoms, molecules, and interactions in greater numbers, we have much greater knowledge about the natural world, but in fact, we still do not know great deal of how it functions. So this is just one more project to try to figure out why the world works the way it does. The fact that there are these unknown genes that clearly have a critically important function, as if you take them away, the cell dies, that needs to be explored – that is an interesting piece of the unknown part of the world that will be good to learn about. A colleague of mine, said something very interesting about health, about human health: “We don’t know why cells die and we don’t know why cells reproduce in an out-of-control manner. But cells reproducing too much or dying to soon – that’s the essence of the human disease.” And fact that we don’t really know how this mechanism works makes it a really worthy target for study.

What is the biggest challenge on our way to build a cell?
So there are the scientific challenges and that is not my area of expertise. The ethical challenges are really profound and I want to spend time thinking about that. Human beings have not made something from nothing. To create a new living organism, to create life out of chemical parts, is an extraordinary leap in human imagination and human power. And that kind of power is non-precedented, because we have made different sorts of moves towards the power and mastery of the universe before, but this is a very different sort of path. This is saying that we could create life, create cellular life – perhaps, going down that road means putting cells together, having them work together, having them differentiate, having them forming organisms… And that power has never been in our hands before. So creating life, not in the usual way that human beings create life, is a startling new idea. And is it startling enough to keep us from doing it? I do not think so, but I think that we have to do it with a great care, with the attention to dynamics of power that it would represent. There is also a theological challenge, because, of course, it is God who creates life and who begins the universe and is known as a Creator – God. So that threading on that territory really changes the relationships to God, to God and gods, and to what it is intended to be, what we are intended to act in world, how we are as humans, so something fundamental about our nature is our fragility, our powerlessness, our limits, and if we take all of that away suddenly with this technology, you are asking the humanity to rethink itself in a profound way and that’s one of the projects that will have to be studied as the larger scientific project goes ahead. If this is successful, one of the other challenges will be the control: who will control the synthetic cells? Is it a for-profit enterprise? How do you make sure that the social goods created by the science are fairly distributed? What system of justice you use to do that? That’s a challenge for genetics in general and synthetic biology just cannot escape the challenge. Finally, people worry about the use of this power for evil, about the capacity to do use it as weaponry, and as a social harm, with such power and such mastery it has. So that has to be – either from scientists themselves or the DIY community itself – ways to regulate it, to make sure that, in fact, the ends and goals of science are the ends and goals that are assets to human beings and to the planet, not ones to be disruptive. And if they cannot be developed internally, there has to be thought about how states, governments, the social groupings then would define these parameters and norms. But someone needs to pay some attention to that. There is nothing ethically impermissible about this project, but there are some things that are certainly ethically challenging.

How would you respond to people who might not be scientists and would be afraid of what we are doing and where the project of building a synthetic cell can take us, especially in relation to the religious beliefs?
Let me first say something interesting about religion. The different religions see and understand the nature differently. For the Jews and many Muslims, the nature is broken, fragile and unredeemed and needs to be made perfect and whole. And that is the human challenge – there is a partnership relationship between the God and the human beings to create the perfect Earth. The nature is fallen, the nature is chaotic, it is fundamentally unjust, and the capacity for the human beings to change that is a big task to Jews and many Muslims – and for some protestants, actually. The Catholics have a different idea – they have the idea that there are the two books of knowledge – the Book of Nature, and the Book of Scriptures. The scripture and nature would provide two sources of information, which is based on the idea that the nature is normative, that the nature has good rules, that the nature, if let alone, will function in harmony. In Pope’s encyclic about the climate change and the global warming, he thinks about the brother and sister relationship between the science and religion, which is very important in Catholic theology. So nature is not broken – nature is whole and we learn from nature, so that is very helpful for a Catholic synthetic biologist because you have the nature, the chemicals that don’t come from nowhere, they come from nature in its most elemental form and you are just recombining them in a different way – kind of like taking the bricks from one house and putting to a new house. I think that task of humanity is to make the world more just. The task of humanity is to learn more about the world, to understand that we are the part of nature and that the nature is not some separate entity, some object that we create. I do think that religion and science go hand-in-hand, as a double-helix of the DNA. There is one problem with this, though, that is to be raised. We have seen the extraordinary destructive capacity of the industrial technology, starting from good ideas of industrializing the world, having steam engines for the transportation, and modern agriculture. But all of that has created this extraordinary carbon disaster and global warming that threatens every scientific enterprise and the most of the human enterprises too. We have to take seriously the problem that people are raising: can you control the technology? Will this have unforeseen and unintended consequences that may have threaten us still further? So I think that built into this is this iterative process, where at each stage it will be important for philosophers and theologians to say how this process is going so far, what are the implications and how to create the national and international dialogue about how this might go right and how this might go wrong. Taking seriously those claims is a part of what we have learned, it is a part of our humility. Science is infinitely progressive – but sometimes there are significant negative consequences that emerge with every human enterprise.

Richard Murray, California Institute of Technology

Highlights:
- The best way to build the cell is the one which actually delivers the desired functioning cell.
- We should build the cell out of the components we understand.
- The biggest challenge on our way to build the cell is the complexity; ability to assemble multiple systems and get them to work together.

What is the best way to build and understand the cell?
I do not know! So I think we’ve got to try stuff and in the end, the best way is the solution that works. I think we want to get something that has minimal functionality – we can define what the minimal functionality is – but it ought to have some cell-like properties; probably be encapsulated, probably have some inputs and outputs, probably have some actions or decisions, or something that happens – and we have to try different things and see what works.

How would you discover the function of the essential unknown genes? Is it even important?
I do not know if it is important or not – I mean, I want to get things that work and I am happy to use genes we understand how they work as part of that, if there is a function that we cannot implement, because we cannot find the protein or biological component that does what we need, we might need to go to discover more. But my kind of gut feeling is that we should build things out of components that we understand, and we probably have enough components that we understand to build the synthetic cell. So that is kind of where I am right now.

What is the biggest challenge on our way to build a cell?
There is not the biggest challenge – there are lots of challenges. I think what is hard about the complex systems is that you have to solve many, many problems and get them all to work and then you’ve got to put them all together and still have them working, so the complexity is the big challenge maybe, or organizing complexity, managing complexity, wrangling complexity – it is probably what ties together all those pieces. But I do not think that there is this sort of “if we solve this problem, we are done.” It is hard to get containers that do all we need, it is hard to get things that go inside the containers, things that make decisions, it is hard to get inputs, it is hard to get outputs, it is hard to get energy going – all of this have interesting problems and I think what you see in the most engineering disciplines is that within each subdomain, there are the hardest problems that subdomains have to solve – probably top ten hardest problems – in order to get something that the other people can use.