So for this part of the talk we're going to talk about synthetic biology.

I
described before how cloning was carried out using the classic molecular biology
and to some extent synthetic biology came to replace that. While in the past
biology started with a complex system and as many parts were taken

away, so that you had this minimal complexity system, and then studied. That's how
biology has worked for centuries. And it's a real top-down approach. So you make
the system as simple as possible, and then you study it. Synthetic biology has
proposed something different. It has proposed that maybe we should be able to study
the individual parts, the individual pieces of DNA, of proteins, and build
complexity from that. In reality, it's not that big a difference in how the
approaches take place, but it has a big impact on the philosophy of the field
itself. So BioBricks is an example of that.

You can do your cloning in a normal classic way, And biobricks remain, to some
sense, a classical molecular biology approach. But what has been introduced here
was the idea of robustness, the idea of modularity, the idea of reproducibility. So
by using that context that does not change as you assemble DNA, the idea was to
make biology more reproducible. But in trying to do so, you actually need more
language because DNA is not defined enough. And this is one of the initiatives that
have begun to try and standardize the language, because if you standardize the
language, even down to the graphs, even down to the glyphs you use to describe a
system, they become reproducible.

is easier to communicate once you learn the system. And this common language is
represented through SBOL, so the Synthetic Biology Open Language. And the idea here
is that for each part of DNA, you gain a symbol. So in here, you can see the arrow
for promoter. So all promoters are represented in the same way. And through that,
you can then represent a circuit. You can represent a protein being expressed. So
in this case, you have GFP being expressed under the control of this promoter,
which we'll see much later that is a constitutive promoter. It also gives you
information. You can highlight that you have an RBS. And if you have a database,
you can highlight down to which RBS you're using. And of course, you highlight the
terminator. So that kind of language is very easy, is very useful to describe a
biological system.

So, for instance, we mentioned in the previous section about the PET vector and
how the sequence you have, the promoter, the lack operator, the RBS, these internal
tags, sort of his tag, T7, his tag, and a terminator. and you can represent that
with sball the same way so you have there your promoter your operator your
individual cut sites rbs and here are the three internal tags that starts at the t7
throne and his tag and here the his tag towards the end you can also highlight
where the termination of translation occurs, and the termination of transcription.
That makes it easier to express what you have at the DNA level, but without having
to provide the sequence. And that brings us back to the very first slide, where I
say the structure matters.

And here I want to really explore what I mean by it depends on your expression
strategy. So, for instance, if you want to clone your gene into PET28, there are,
of course, multiple ways of doing it.

So, you must consider your end product and how you want to do it. So, in this
example, you have here the PET28 backbone ready. if you want if you have a gene for
instance that has its own terminator translation you could for instance try and
clone it in the nde and sal1 sites so this is the nde one here sal1 further down
and the gene can be inserted there the consequence of inserting there is of course
you have your his tag still there, you have your thrombin site still there,
preceding your gene. So you have a different protein than the one you had coded
here, because you have these additional tags. So again, if you look back at the
previous slide, where the structure matters, of course,

change in the sequence of the protein may affect the structure. But this is simply
to highlight that you had that choice, and of course this his tag is not used,
because the translation has already stopped. But if you were to clone it, NCOXH01,
this initial site immediately downstream from the RBS is your NCO1. and one of the
sites much later at the XHO1. And the result is a very different protein. So of
course, the core of the protein is exactly the same. But now you have a little link
and the final histag. This little link is an artifact. It really emerges from the
sequence you have in the system. So if we go back here, if you can see there, the
XO801 site, and to use that as a cloning site, if your gene is not bringing a stop
translation signal, means that the transcription is going to continue, the
translation is going to continue, and you then have your histag after a very short
linker. And while in the lab these differences may not be big, may not even be
significant, they can amount to significant changes in the patient. Therefore, it
is very important to understand how cloning is done because the small subtle
differences in cloning can lead to big differences in the product. So again, it's
what I promised I would say every lecture, the process determines the product. So
what does it look like? I mentioned, I gave an example in the first lecture,

how you're having three fragments and you're trying to bring everything into a
single vector. What it looks like, you had to choose your enzymes, you had to find
compromises. What BioBrick changes is what I'm trying to show here. so you have
there the old example where you can clone in a certain point but now if you have a
database of parts if you have a kind of a series of rbs is a series of operators
promoters genes of interest tags and terminators you can start doing the biobrick
assembly and bringing each part into composite parts and then assembling those
composite parts into newer parts until you have your circuit so again here you have
the promoter which is for regular for initiation of transcription you have your
operator which again is controlling transcription you have your rbs which is
beginning kind of is the control of translation then leads to your gene of interest
being translated with a tag that we'll see later why you may want to use a tag and
then termination of translation followed by termination of transcription and
because it's biobricks you know and you can predict exactly which little scars are
going to be between each one of those parts because this is reproducible because
this is the only way you end up being able to operate this becomes standardized and
that's very important because now it becomes a lot more reproducible but while i
present you biobricks they are other standards and for instance this

is the european standard but again where you have certain parts of the plasmid
which are present everywhere so here you can see the antibiotic resistance here you
can see the replication although the the saver can sometimes have more than one
replication so that it can go into multiple hosts. And where it says cargo, that
would be your gene of interest, protein of interest. But ultimately, between those
two is where you would build your circuit. And because of the standardization, once
you characterize these plasmids, once you use them, you start to accrue knowledge.
So this is another powerful tenet of synthetic biology. as you acquire more
knowledge because now everything is standardized you can accelerate discovery and
the impact of that

you can see for instance in Ginkgo Bioworks so this is a company that operates on
a synthetic biology promise they want to assemble DNA fast robustly and test it to
develop new products be it fragrance, fuel or new materials a couple of years ago
you see that 2019 they were worth four billion dollars they've now been floated and
they've already exceeded 15 billion dollars so synthetic biology is definitely
growing very fast and it will dominate biotechnology in the coming years and with
that we mentioned

kind of we come back to the the three things we do as we move across the dna the
central dogma so you have to maintain dna you have to control transcription you
have to control translation and how does it look in a normal typical
bacterioplasmid so i try here to summarize

how those three aspects come together so a plasmid always needs an origin of
replication Coly-1 being one of the most common ones among the plasmids we use
normally. That allows the plasmid to replicate. You also need an antibiotic
selection marker, which of course needs its promoter, RBS, and terminator, so that
you can transcribe and translate the selectable marker. So you know that if your
cells survive in the presence of kanamycin, it contains this plasmid. So that's the
key maintenance of the plasmid. And then you go into the transcription regulation.
So promoters, operators, terminators. So in this particular case, I have here a T7
promoter regulated by the LAC operators that will then translate this sort of
tagged gene of interest until it terminates. But within that transcription, which
again, this is all being defined at the DNA level, but of course, transcription
will make it into RNA, and it's the RNA that then contains the regulatory
sequences. But that regulatory sequence is, of course, encoded in the DNA itself as
well. But then you have the translational regulation. So be it the RBS, the
initiation site, the initiating methionine, or the stop codons at the very end. And
that sort of is the control of translation. What we'll come back to is this, this
impact on the cell economy. Because to express that protein, there will always be a
metabolic cost. And the field of biotechnology has really progressed towards trying
to understand that cost and what it means when you try to scale up the production.