Biodegradable Plastic: Engineering a Bioplastic Producing E. Coli

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00:00:11.18 Hi there, my name is Cordelia.
00:00:13.10 Myself and my colleague, Nico, will be introducing our
00:00:15.26 synthetic biology project,
00:00:17.13 which is to engineer a bioplastic-producing
00:00:19.13 E. coli cell factory.
00:00:20.26 And the problem with most plastic production
00:00:23.26 currently is that plastic is derived from crude oil,
00:00:26.10 which is then refined into petroleum.
00:00:27.25 And when you're obtaining this crude oil from the ground,
00:00:29.28 you often end up with horrible environmental consequences.
00:00:32.10 Such as this oil spill here.
00:00:34.05 Another problem is, because plastic
00:00:36.14 is so cheap, people often throw it away without consequence
00:00:39.10 and so you end up with these enormous landfill sites
00:00:41.25 which are just full of this plastic,
00:00:43.18 which won't break down for up to a thousand years.
00:00:45.07 In addition, sea birds and animals will
00:00:49.10 also pick up this plastic and ingest it,
00:00:51.11 and it then sits in their gut where it
00:00:53.02 can't break down, and often this leads
00:00:55.00 to death. As you can see with this unfortunate bird.
00:00:56.24 Another problem is that, plastic actually
00:01:00.12 breaks down over time into these
00:01:01.29 microplastics, which end up accumulating
00:01:03.28 in the sea. And you can see here the currents
00:01:05.25 have actually caused these great big
00:01:07.14 garbage patches in the Pacific Ocean.
00:01:09.05 So, a solution we have is to produce
00:01:12.22 this bioplastic out of biomass.
00:01:15.02 So, biomass is any kind of plant material
00:01:18.10 really in this situation, particularly lignocellulose,
00:01:21.18 which is a very hard to break down product.
00:01:23.10 And in this case, you can break it down into sugars,
00:01:26.13 which is then a carbon source for our engineered
00:01:28.06 bacteria. And these engineered bacteria can then
00:01:31.02 produce a biodegradable bioplastic.
00:01:32.26 And the good thing about this is that it's actually cyclical,
00:01:35.13 so it's a renewable cycle where environmental bacteria
00:01:38.26 can then break down this biodegradable plastic,
00:01:40.23 release carbon dioxide, which is then sequestered by
00:01:43.07 the plant, and you can feed the whole process
00:01:44.28 again. Now it's time for the practical
00:01:47.01 part of our project. How are we going to implement
00:01:50.04 bioplastic production in a bacterium?
00:01:52.11 We chose to work with PHB,
00:01:55.19 which stands for poly(3-hydroxybutyrate),
00:01:58.13 as you can see it right here.
00:01:59.23 PHB is actually a natural storage polymer
00:02:02.29 in many microorganisms. However, we
00:02:05.11 would like to work with E. coli and E. coli
00:02:07.11 does naturally not produce PHB.
00:02:09.08 But E. coli is a really nice microorganism to work
00:02:12.16 with in the lab, as it's very fast growing,
00:02:14.22 and a lot of molecular biology and synthetic biology
00:02:17.04 tools are available for E. coli.
00:02:19.23 E. coli's natural central metabolism can convert glucose
00:02:24.00 up here into the common precursor, acetyl CoA.
00:02:27.22 And acetyl CoA can actually be a precursor for
00:02:30.23 PHB production. So we can add a pathway
00:02:33.17 that converts acetyl CoA to PHB.
00:02:36.02 You can see a pathway here.
00:02:38.06 We decided to work with the pathway of
00:02:41.02 Cupriavidus necator, which is a natural organism
00:02:43.22 making PHB. The pathway consists of three enzymes
00:02:48.01 doing the whole conversion. In addition to engineering
00:02:52.01 this pathway, we like to improve the PHB production
00:02:54.18 further by testing some E. coli knockout
00:02:57.23 mutants. The first knockout mutant which
00:03:00.25 we are testing is this knockout here,
00:03:04.18 of this enzyme which is one of the first enzymes
00:03:08.01 in glycolysis. So basically blocking all the flux
00:03:11.01 into glycolysis and forcing it in other ways.
00:03:13.06 In addition, we're testing a single knockout
00:03:16.17 mutant in the pentose phosphate pathway,
00:03:19.02 which basically means no flux is going into this pathway.
00:03:22.07 A third very interesting mutant we think
00:03:26.11 also based on metabolic modeling would be
00:03:29.01 knocking out the Pta, Pta is the first gene
00:03:33.13 to what's acetate fermentation, converting acetyl CoA
00:03:36.16 in the end into acetate. And we think that this fermentation
00:03:40.00 pathway takes a lot of the acetyl CoA, which we would
00:03:42.10 actually like to force into the PHB production.
00:03:45.08 So, we'd like to test all those three and
00:03:49.23 adding the pathway. So it's time to get into the lab
00:03:52.06 and get some bacteria going and isolate
00:03:54.16 some DNA.
00:03:57.02 But wait! Before we continue, we have to know
00:04:19.08 how are we going to introduce these three genes,
00:04:22.07 which encode those three enzymes for PHB production
00:04:24.29 into E. coli. There are actually multiple approaches
00:04:28.06 possible, and we chose to investigate two approaches.
00:04:31.02 The first approach we're looking into is
00:04:34.02 introducing the three genes on a plasmid.
00:04:36.28 A plasmid is a short circular DNA that replicates
00:04:40.11 itself in a cell like E. coli. And there will be multiple
00:04:44.07 copies of this plasmid, so you get a relatively
00:04:46.13 high dosage of the gene, which might be interesting
00:04:50.04 for getting a lot of production. On the other hand,
00:04:52.17 it might be a high metabolic stress for the cell.
00:04:54.29 In addition, plasmids have a small drawback in that
00:04:59.00 they're not always stable in a cell.
00:05:00.29 And one of the ways to keep them in is
00:05:03.14 actually having to add antibiotic all the time,
00:05:06.08 as there's an antibiotic resistance marker
00:05:08.04 on the plasmid. As an alternative approach,
00:05:11.18 we tried to introduce the three genes
00:05:14.10 for the bioplastic production and an antibiotic marker
00:05:17.12 onto the chromosome of E. coli,
00:05:19.05 which is potentially way more stable.
00:05:20.25 Therefore, we use a tool called transposons.
00:05:23.24 Transposons are natural genetic elements
00:05:27.01 and they can also be used as a synthetic biology
00:05:29.18 tool to introduce stuff on a chromosome.
00:05:32.10 They have the property that they introduce
00:05:35.05 genes on a random place in the chromosome, which
00:05:37.23 might be interesting because you might get many
00:05:40.13 different expression levels dependent on the
00:05:42.05 place of the chromosome where it gets integrated.
00:05:44.13 And in addition, it seems to be really stable.
00:05:46.29 So, we're also going to try that.
00:05:49.08 So, we managed in the mean time to get the plasmids
00:05:54.01 into our E. coli strains by transformation.
00:05:57.16 We didn't manage completely yet to
00:06:00.07 get the transposons into the chromosomes.
00:06:02.18 So, more about that later.
00:06:04.28 But we already have some data on the plasmids
00:06:07.17 expressions of the genes.
00:06:09.13 If we express those genes from the plasmid
00:06:12.22 we seem to get PHB and we found that out
00:06:15.19 by using an indicator molecule, which
00:06:17.16 is called Nile red. As you can see here
00:06:20.04 if we add Nile red to our cell cultures, they get very red
00:06:22.24 if we have the PHB producing strains.
00:06:25.29 And this looks nice, but of course we'd like to quantify
00:06:30.05 how the different mutants and the wild type produce
00:06:33.05 PHB. So that's why we went to measure
00:06:36.02 in a 96-well plate the fluorescence of this
00:06:39.05 red compound. After putting this plate in a
00:06:42.10 plate reader, we get the data on the PHB production.
00:06:46.24 And as you can see, this is quite interesting.
00:06:50.13 All the strains produce some PHB, but the Pta
00:06:54.08 knockout, if you remember that was the one
00:06:56.12 going into the acetate fermentation, seems to produce
00:06:59.11 far more PHB than all of the other knockouts.
00:07:01.16 So this is a really interesting strain
00:07:03.23 to continue with. In addition of course
00:07:06.29 we'd like to see what transposons on the chromosome would do.
00:07:09.23 We didn't manage to do it ourselves completely yet,
00:07:13.06 it's not practical, but there's already some data
00:07:16.12 available from literature on this.
00:07:18.13 Last year, it was published that production from
00:07:21.24 the chromosome in the same strain actually we are using
00:07:25.13 gives a bit lower production than optimal production
00:07:28.10 with a knockout, the one we used actually,
00:07:31.02 gives a bit lower production than that one,
00:07:33.19 about 78%. That's probably because there
00:07:37.12 is lower gene dosage, having one integration
00:07:39.24 into the chromosome instead of all the plasmids
00:07:42.11 in your cell. However, in this paper they also showed
00:07:45.16 that the cell with the chromosome knockin has
00:07:49.01 a way better growth than the other cell.
00:07:51.16 So, it might still be an advantage
00:07:53.29 to use transposons.
00:07:55.22 Of course it's nice to see red colors
00:07:58.11 in cells, but in the end, we would like to see
00:08:00.07 real PHB. So, we had to do some more real work.
00:08:02.26 We had to use our strain, the one with the plasmid
00:08:06.02 to purify the PHB using extraction.
00:08:09.13 And we succeeded.
00:08:11.20 And here we have our overnight culture of bacteria
00:08:15.22 in an Erlenmeyer flask, and we then
00:08:17.22 freeze dried this in a lyophilizer and our next step was
00:08:21.18 to do a solvent extraction, and this was a two phase
00:08:24.00 process using acetone and chloroform,
00:08:26.02 and we then were able to precipitate out
00:08:28.28 our plastic, our final product, poly(3-hydroxybutyrate).
00:08:32.11 In conclusion, we have found that PHB
00:08:35.10 production in E. coli is feasible, however,
00:08:38.04 it's a very expensive process.
00:08:39.11 And in our pilot study, we found that we only
00:08:41.14 got 1% yield from our biomass. And so this obviously
00:08:44.19 isn't particularly viable as a commercial
00:08:46.16 enterprise. So what we actually plan to do is
00:08:49.08 to optimize the strains and perhaps look at different
00:08:51.14 host organisms, maybe to use yeast instead.
00:08:53.24 The high cost we found was mostly associated
00:08:56.16 with the acetone and the chloroform extraction method
00:08:59.06 we used, so we could look at using alternatives.
00:09:02.01 The problem with these solvents is that they're very difficult
00:09:04.05 to get rid of. In the future, perhaps we could use
00:09:07.10 synthetic biological approaches to
00:09:09.05 develop new bioplastics and perhaps even
00:09:11.19 replace many of the conventional plastics that we use.[00:09:14.00

This Talk

Audience:

Researcher

Recorded: June 2015

Talk Overview

Most plastic is derived from crude oil. Synthesis of plastics and their accumulation in oceans and landfills lead to bad environmental consequences. One solution to this problem is to create biodegradable plastics from biomass using bacteria. These bacteria can be engineered into plastic producers using synthetic biology tools. The Synthetic Biology in Action course participants tried this approach, and, in this video, they explain the experiments that led to the production of a biodegradable plastic.