Team:Slovenia HS/old
Welcome to Slovenia HS's team Wiki! |
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ABOUT USWe are the first high school team from Slovenia to compete at iGEM and are very excited to face the challenges synthetic biology poses. The team is composed of eight high school students attending seven different secondary educational institutions across Slovenia. Our research is performed at the Laboratory for Environmental Sciences and Engineering, National Institute of Chemistry, Slovenia and at the Chair of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana under supervision of knowledgeable and encouraging mentors.
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WHAT WE DOWe have decided to tackle a
very imposing
ecological problem that is becoming more and more threatening due to
fossil fuel
depletion and a steady growth of energy consumption. Fossil fuels, such
as oil
and gasoline, have gained popularity in the beginning of the 20th
century,
following the industrial revolution. It is estimated that we have spent
between
100 and 135 billion tons of oil since 1850 and the demands are still
increasing. Fossil fuels are used in cars, airplanes and other
vehicles, to
power electricity plants, to heat our homes and to make many everyday
products,
such as medicines, cosmetics, plastics and synthetic fabrics. Our
society is
largely dependent on them, but fossil fuels take millions of years to
form and
are therefore non-renewable resources. According to some projections,
we only
have enough oil for the next 40 years, so it is becoming increasingly
necessary
to find an alternative method of obtaining fuels. After a thorough discussion and
literature
survey, we have thus decided to make use of butanol. Research has
already shown
that because of its long chain and consequent nonpolarity, butanol can,
amongst
other uses, replace gasoline in internal combustion engines (it can be
used in
existing diesel engines without considerable modifications of the motor
system). In nature, many organisms have
been proven to
be able to produce butanol (n-butanol or 1-butanol) by converting
glucose into
acids and then converting acids into alcohols. This organisms, mostly
bacteria
of the Clostridium genus (Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium
saccharoperbutylacetonicum),
however, have complex metabolism, slow conversion rate and are often
hard to
grow in laboratories or for industrious use. For these and other
reasons, they
are unsuitable for larger butanol production. On the other hand,
bacteria E.
coli have relatively simple and strikingly fast metabolism, already
researched
and utilized mechanisms for genetic manipulation and are relatively
easy to
grow and cultivate, making them the perfect laboratory and industrial
organisms. We have sought help from the
Laboratory for Environmental
Sciences and Engineering at the National Institute of Chemistry in
Ljubljana,
as they have been engaged in advancing processes that enable conversion
of
waste and other renewable raw materials into energy (e.g. biogas to
syngas
transformation, bio-oil production through pyrolysis of waste etc.) for
a long
time now, and have recently also developed a process for the
biotechnological conversion
of biodegradable waste into hydrogen by means of anaerobic mixed
cultures grown
at special fermentation conditions. During this process a series of
intermediate products is produced, amongst others are high
concentrations of butanoic
acid. We had the idea of turning this organic acid into butanol, which
is
hydrophobic and can be separated from water medium by basic
concentration and
decanting processes. That’s why we have
set on the path to modify
the E. coli in such a way, to
enable
transformation of butanoic acid to butanol. To aid in the process, we
will also
be using glycerol as a co-substrate, which can be readily obtained in a
suitable form as a side product of biodiesel production processes. We will thus genetically
manipulate the E. coli bacteria
into performing only
the second phase of butanol production found in Clostridium bacteria
(acids to
alcohols conversion). By using biodegradable waste as substrate we
intend to
fully make use of all of the components involved in this processes
(anaerobic
microbial production of hydrogen), while subsequently (E.
coli fermentation process) also producing the much-needed
biofuel, thus enabling a simultaneous efficient and eco-friendly waste
management and energy production process. Our aim is to achieve the
highest
possible yields with our genetically modified in an optimized
bioreactor system. In the first stage the biogas (H2)
will be isolated out of organic waste and suspended solids will be
produced
which may be used as a natural fertilizer. At the same time the
butanoic acid
formed will be redirected in another bioreactor (containing our
modified E. coli) where it will be
converted into
bio-butanol. Up to now, we have cloned three
genes coding
for CTFA, CTFB and BDHB proteins (in PSB1C3 vector), that are
responsible for
butyrate aldehyde to butanol in Clostridium
acetobutylicum. We further intend to make composite
constructs to include
this genes in vectors with promotors and ribosomal binding sites to
allow for
expression of these genes in E. coli. In the biotechnological
laboratory we have
performed an initial screening of growth conditions, such as butanoic
acid
concentration and butanoic acid / glycerol molar ratio, as well as
comparing
the growth in the presence and absence of oxygen. We are working with
two
negative controls, (i) E. coli
strain
Dh5 alpha and E. coli strain Dh5 alpha with PSB1C3 plasmid containing
only the
promotor and the ribosomal binding sites (i.e. with no genes included),
to
provide proof E. coli does not inherently convert butyric acid into
butanol. We
intend to perform similar tests with our final microorganism
– modified E. coli
and provide proof of concept for the said fermentation in a bench-scale
laboratory bioreactor. We are also looking for other teams with similar projects for possible collaborations, so if you are interested, we encourage you to contact us! |
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READ MORE ABOUT OUR PROJECT |