Hi there, my name is Cordelia.
Myself and my colleague, Nico, will be introducing our
synthetic biology project,
which is to engineer a bioplastic-producing
E. coli cell factory.
And the problem with most plastic production
currently is that plastic is derived from crude oil,
which is then refined into petroleum.
And when you're obtaining this crude oil from the ground,
you often end up with horrible environmental consequences.
Such as this oil spill here.
Another problem is, because plastic
is so cheap, people often throw it away without consequence
and so you end up with these enormous landfill sites
which are just full of this plastic,
which won't break down for up to a thousand years.
In addition, sea birds and animals will
also pick up this plastic and ingest it,
and it then sits in their gut where it
can't break down, and often this leads
to death. As you can see with this unfortunate bird.
Another problem is that, plastic actually
breaks down over time into these
microplastics, which end up accumulating
in the sea. And you can see here the currents
have actually caused these great big
garbage patches in the Pacific Ocean.
So, a solution we have is to produce
this bioplastic out of biomass.
So, biomass is any kind of plant material
really in this situation, particularly lignocellulose,
which is a very hard to break down product.
And in this case, you can break it down into sugars,
which is then a carbon source for our engineered
bacteria. And these engineered bacteria can then
produce a biodegradable bioplastic.
And the good thing about this is that it's actually cyclical,
so it's a renewable cycle where environmental bacteria
can then break down this biodegradable plastic,
release carbon dioxide, which is then sequestered by
the plant, and you can feed the whole process
again. Now it's time for the practical
part of our project. How are we going to implement
bioplastic production in a bacterium?
We chose to work with PHB,
which stands for poly(3-hydroxybutyrate),
as you can see it right here.
PHB is actually a natural storage polymer
in many microorganisms. However, we
would like to work with E. coli and E. coli
does naturally not produce PHB.
But E. coli is a really nice microorganism to work
with in the lab, as it's very fast growing,
and a lot of molecular biology and synthetic biology
tools are available for E. coli.
E. coli's natural central metabolism can convert glucose
up here into the common precursor, acetyl CoA.
And acetyl CoA can actually be a precursor for
PHB production. So we can add a pathway
that converts acetyl CoA to PHB.
You can see a pathway here.
We decided to work with the pathway of
Cupriavidus necator, which is a natural organism
making PHB. The pathway consists of three enzymes
doing the whole conversion. In addition to engineering
this pathway, we like to improve the PHB production
further by testing some E. coli knockout
mutants. The first knockout mutant which
we are testing is this knockout here,
of this enzyme which is one of the first enzymes
in glycolysis. So basically blocking all the flux
into glycolysis and forcing it in other ways.
In addition, we're testing a single knockout
mutant in the pentose phosphate pathway,
which basically means no flux is going into this pathway.
A third very interesting mutant we think
also based on metabolic modeling would be
knocking out the Pta, Pta is the first gene
to what's acetate fermentation, converting acetyl CoA
in the end into acetate. And we think that this fermentation
pathway takes a lot of the acetyl CoA, which we would
actually like to force into the PHB production.
So, we'd like to test all those three and
adding the pathway. So it's time to get into the lab
and get some bacteria going and isolate
some DNA.
But wait! Before we continue, we have to know
how are we going to introduce these three genes,
which encode those three enzymes for PHB production
into E. coli. There are actually multiple approaches
possible, and we chose to investigate two approaches.
The first approach we're looking into is
introducing the three genes on a plasmid.
A plasmid is a short circular DNA that replicates
itself in a cell like E. coli. And there will be multiple
copies of this plasmid, so you get a relatively
high dosage of the gene, which might be interesting
for getting a lot of production. On the other hand,
it might be a high metabolic stress for the cell.
In addition, plasmids have a small drawback in that
they're not always stable in a cell.
And one of the ways to keep them in is
actually having to add antibiotic all the time,
as there's an antibiotic resistance marker
on the plasmid. As an alternative approach,
we tried to introduce the three genes
for the bioplastic production and an antibiotic marker
onto the chromosome of E. coli,
which is potentially way more stable.
Therefore, we use a tool called transposons.
Transposons are natural genetic elements
and they can also be used as a synthetic biology
tool to introduce stuff on a chromosome.
They have the property that they introduce
genes on a random place in the chromosome, which
might be interesting because you might get many
different expression levels dependent on the
place of the chromosome where it gets integrated.
And in addition, it seems to be really stable.
So, we're also going to try that.
So, we managed in the mean time to get the plasmids
into our E. coli strains by transformation.
We didn't manage completely yet to
get the transposons into the chromosomes.
So, more about that later.
But we already have some data on the plasmids
expressions of the genes.
If we express those genes from the plasmid
we seem to get PHB and we found that out
by using an indicator molecule, which
is called Nile red. As you can see here
if we add Nile red to our cell cultures, they get very red
if we have the PHB producing strains.
And this looks nice, but of course we'd like to quantify
how the different mutants and the wild type produce
PHB. So that's why we went to measure
in a 96-well plate the fluorescence of this
red compound. After putting this plate in a
plate reader, we get the data on the PHB production.
And as you can see, this is quite interesting.
All the strains produce some PHB, but the Pta
knockout, if you remember that was the one
going into the acetate fermentation, seems to produce
far more PHB than all of the other knockouts.
So this is a really interesting strain
to continue with. In addition of course
we'd like to see what transposons on the chromosome would do.
We didn't manage to do it ourselves completely yet,
it's not practical, but there's already some data
available from literature on this.
Last year, it was published that production from
the chromosome in the same strain actually we are using
gives a bit lower production than optimal production
with a knockout, the one we used actually,
gives a bit lower production than that one,
about 78%. That's probably because there
is lower gene dosage, having one integration
into the chromosome instead of all the plasmids
in your cell. However, in this paper they also showed
that the cell with the chromosome knockin has
a way better growth than the other cell.
So, it might still be an advantage
to use transposons.
Of course it's nice to see red colors
in cells, but in the end, we would like to see
real PHB. So, we had to do some more real work.
We had to use our strain, the one with the plasmid
to purify the PHB using extraction.
And we succeeded.
And here we have our overnight culture of bacteria
in an Erlenmeyer flask, and we then
freeze dried this in a lyophilizer and our next step was
to do a solvent extraction, and this was a two phase
process using acetone and chloroform,
and we then were able to precipitate out
our plastic, our final product, poly(3-hydroxybutyrate).
In conclusion, we have found that PHB
production in E. coli is feasible, however,
it's a very expensive process.
And in our pilot study, we found that we only
got 1% yield from our biomass. And so this obviously
isn't particularly viable as a commercial
enterprise. So what we actually plan to do is
to optimize the strains and perhaps look at different
host organisms, maybe to use yeast instead.
The high cost we found was mostly associated
with the acetone and the chloroform extraction method
we used, so we could look at using alternatives.
The problem with these solvents is that they're very difficult
to get rid of. In the future, perhaps we could use
synthetic biological approaches to
develop new bioplastics and perhaps even
replace many of the conventional plastics that we use.[00:09:14.00