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Plastic degradation and Hydrogen Production using

Ideonella sakaiensis PETase via 3A Assembly in
Escherichia Coli

Experiment Findings · March 2019

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Sean Brown
University of Maryland, Baltimore County
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 Plastic degradation and Hydrogen Production using Ideonella sakaiensis PETase via
3A Assembly in Escherichia Coli
Abstract
Here we present our work done throughout the spring 2019 semester in
BIOL 306L to create a synthetically modified strain of E. coli that will
be able to degrade plastic as well as produce hydrogen on an industrial
scale. Plastic has been an attractive material for many years due to its
durability and inexpensiveness. However, because of its durable nature,
plastic is very difficult to degrade, therefore leading to plastic littering
and pollution. Hydrogen production has also gained popularity for its
ability to store and deliver energy in a cleaner, usable form. However,
current hydrogen production methods involve the use of fossil fuels that
can cause harm to the environment. We have successfully obtained
DNA from E. coli with the plastic degrading enzyme, PETase, and
created the ideal E. coli plasmid to have the enzyme inserted into using
BioBrick 3A Assembly methods.
Introduction
Hydrogen is the most abundant element in the observable universe, yet
gaseous hydrogen makes up a miniscule amount of the composition of our
atmosphere. Hydrogen gas is beginning to gain ever increasing market
usefulness. The United States alone is now producing upwards of 10 million
metric tons of hydrogen gas per year (2). Most of this production is achieved
through the use of fossil fuels, such as natural gas and coal (1). Not only is
this method inefficient and environmentally unfriendly, but it is expensive as
well. Plastics are also becoming an ever increasing problem. Polyethylene
terephthalate (PET) accounts for a vast amount of plastics produced,
including ~60% of all plastic bottles(6). We hypothesized if there was a
biological machine that could produce sufficient hydrogen at the same time
of using PET as an active carbon source, we could produce machinery that
will provide a constant source of clean hydrogen gas, as well as digesting
PET in the process. We proposed to alter the genome of Escherichia coli,
strain K-12, via 3A assembly with BioBrick parts from iGEM, the PETase
gene from Ideonella sakaiensis, the gene that biochemically synthesizes the
enzyme PETase which breaks down PET into a digestible carbon source, and
via CRISPR Cas 9 to functionally knock out the LDH pathway, which
produces lactate and consumes 2[H], and the ADH pathway, which produces
ethanol and consumes 4[H] (6). 3A assembly is a methodology where
ligation of inserts occur from plasmids with differing antibiotic resistance
into a plasmid with an even different plasmid with antibiotic resistance. This
allows for a screening method that assures that colonies that grow are the
ones with the gene of interest attached, because of selective pressure from the
antibiotic on the plate. BioBrick parts are a set of standard DNA parts with
known prefixes and suffixes (EcoRl, Xbal, Spel, and Pstl restriction sites).
CRISPR cas9 is a methodology of knocking out pathways via the cas9
enzyme and guide RNA effectively cutting out the DNA that codes for
enzymes required for that pathway (1). Knocking out the LDH and ADH
pathways effectively frees up 6[H] and allows for a higher pyruvate
concentration, driving the formate pathway which produces the diatomic
hydrogen and carbon dioxide gas (see fig. 4)
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Sean M. Brown & Tammy T. Tran
University of Maryland Baltimore County, Department of Biological Sciences
Methodology Results Conclusion
Step 1: Create an ideal plasmid using BioBrick parts from iGem registry including the
promoter, ribosome binding site (RBS), and reporter gene. Parts introduced into E. coli by Throughout our studies, we have data that supports that we
bacterial transformation using zippy cells. Plasmids cut by restriction digest enzymes EcoRl
successfully created and obtained DNA from our ideal reporting
(E), Xbal (X), Spel (S), and Pstl (P). Parts put together by 3A Assembly ligation using T4 DNA
ligase. DNA obtained through boiling miniprep. plasmid (BBa_J23119 + BBa_B0034 + BBa_K592009) (see Fig. 5
and Fig. 6). Additionally, we have data to support that we have
successfully obtained DNA from the plasmid that has the codon
optimized PETase plasmid (see Fig. 7). Via 3A assembly we have
almost created an E. coli culture that produces this enzyme while
being reported via a blue chromoprotein (BBa_K592009).
Fig. 5 Agarose Gel, lane 1 - 1kb ladder, lane 2 -
Ligation of full plasmid (~2600 bp), lane 3 - Ligation
Fig. 1- Proposed concept map of plasmid B (~2600bp)
of construction of BioBrick
parts via 3A Assembly. J23119-
Constitutive promoter, B0034-
RBS, K592009- Blue
chromoprotein reporter gene. Future Outlook
Fig. 6 LB Plate of Final BioBrick™ We have successfully obtained DNA from both the PETase and the
Plasmid. Plated on LB with tetracycline ligated plasmid. With this being said, we still need to rapid transform
antibiotic. Healthy colonies indicates them into a BL21(DE3) strain, where both plasmids would be present
successful ligation of
BBa_J23119+BBa_B0034+ due two selective pressure of antibiotics. After successfully
BBa_K592009 plasmid transforming both plasmids, we plan on testing expression levels via
induction of IPTG. Next, we will test enzyme activity with an ELISA
Assay. After we have evidence for expression and activity, we plan on
Step 2: Introduce plastic degrading enzyme- PETase.
using CRISPR-Cas9 for the knockouts of the ADH and LDH
pathways. Post knockout, we plan on doing tests and optimizations for
the activity levels of the PETase enzyme, as well as a practical
application to determine if the modified cells can live off of PET as a
main carbon source. After all optimizations and testings, we plan on
engineering a temperature and pH controlled generator, that will
Fig. 2- PETase optimized plasmid effectively produce hydrogen gas at the expense of plastic, or other
pET21b(+)-Is-PETase-W159H-S238F.
Source: Addgene carbon sources. This will be marketed towards energy companies that
have an interest in hydrogen fuel cell production, such as Shell.
Fig. 7 Agarose Gel, lane 1 - 1kb ladder, lane 2 Purified PETase
culture A DNA (~6200 bp), lane 3 Purified PETase culture B DNA
(~6200 bp)
Step 3: Perform CRISPR-Cas9 to knock out competing pathways.
Citations Acknowledgements
1. CRISPR Guide. (n.d.). Retrieved from https://www.addgene.org/crispr/guide/
2. Fact of the Month May 2018: 10 Million Metric Tons of Hydrogen Produced Annually in the
United States. (n.d.). Retrieved from We would like to thank Dr. Cynthia Wagner for guiding us through this
https://www.energy.gov/eere/fuelcells/fact-month-may-2018-10-million-metric-tons-hydrogen research. Additionally, we would like to thank the members of iGem for
-produced-annually-united-states
3. Fan, Z., Yuan, L., & Chatterjee, R. (2009). Increased Hydrogen Production by Genetic allowing an open source biological parts library to be used. Lastly we would
Engineering of Escherichia coli. PLoS ONE,4(2). doi:10.1371/journal.pone.0004432
4. Hydrogen Production. (n.d.). Retrieved from
like to acknowledge the Biology department at UMBC and all the staff who
https://www.energy.gov/eere/fuelcells/hydrogen-production worked behind the scenes to make this project possible.
5. Hjersing, C. (2011). Hydrogen production in Escherichia coli – Genetic engineering of the
formate hydrogenlyase complex. Linköpings Universitet Institutionen För Fysik, Kemi Och
Biologi.
6. Sojobi, A. O., Nwobodo, S. E., & Aladegboye, O. J. (2016). Recycling of polyethylene
terephthalate (PET) plastic bottle wastes in bituminous asphaltic concrete. Cogent
Engineering,3(1). doi:10.1080/23311916.2015.1133480
Fig. 3 CRISPR-Cas9 methodology. Source: Addgene Fig. 4- E. coli metabolic pathways of interest. Red ‘X’s indicate
planned knockout with CRISPR Cas 9.