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.
