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Commit 4c01515c authored by Yangyang Kong's avatar Yangyang Kong
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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page_content %}
{% block header_content %}
<img class="sub-header-logo" src="https://static.igem.wiki/teams/4515/wiki/banner.jpg" />
{% endblock %}
<div class="row mt-4">
<div class="col">
<div class="bd-callout bd-callout-info">
<h4>Silver Medal Criterion #1</h4>
<p>Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.<p>
<p>If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a> for your team to be eligible for this criteria.</p>
<hr>
<p>Please see the <a href="https://competition.igem.org/judging/medals">2022 Medals Page</a> for more information.</p>
{% block page_content %}
<div class="sub-page-bg">
<div class="sub-page-content">
<div class="content-title">Engineering</div>
<div class="title blue-title">Introduction</div>
<div class="article-content margin-bottom-16">
N-butanol, an important chemical raw material, is expected to become one of the new generations of biofuels. At
present, the domestic industrial synthesis of N-butanol mainly adopts the low-pressure carbonyl synthesis method,
from the production process, propylene, CO, H2, and carbonyl are the main production materials. However, this
production method relies on non-renewable petroleum products as essential raw materials, which is not friendly to
the environment. Therefore, it is necessary to design an environmentally friendly production method for N-butanol
to meet the demand of environmental protection.
</div>
<div class="article-content ">
It has been found that N-butanol can be synthesized naturally in Clostridium, but the tolerance of Clostridium
bacteria is not good enough for large-scale production. Recently, Lactobacillus Brevis with better N-butanol
tolerance has been isolated by researchers. Therefore, this project develops an engineering N-butanol-producing
bacteria to improve the yield of N-butanol, meet the needs of industrial production, and lay a foundation for
subsequent improvement.
</div>
<div class="title blue-title">How we design our plasmid</div>
<div class="article-content ">The N-butanol pathway we developed according to the pathway in Lactobacillus Brevis
ATCC824. To achieve this, we designed the DNA sequences of thlA, crt, hbd, and ter gene into the ApaI and BglII
sites of the pIB184 vector (Figure 1.), and transferred the recombinant plasmid into Streptococcus Brevis ATCC367
for fermentation. </div>
<div class="img-wrap no-margin">
<img class="w-40" src="https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering01.png" />
<span>Figure 1. The map of recombinant plasmid</span>
</div>
<div class="title blue-title">How we build our plasmid</div>
<div class="article-content">To build the plasmid, firstly amplified the thlA, crt, hbd, and ter genes
fragments from the Lactobacillus Brevis ATCC824 genomic DNA (Figure 2). The second step was to obtain the linear
carrier. The third step was to ligate the genes and linearized vector and transfer the ligation product into E.
coli DH5α competent. To build the plasmid, firstly amplified the thlA, crt, hbd, and ter genes fragments from the
Lactobacillus Brevis ATCC824 genomic DNA (Figure 2). The second step was to obtain the linear carrier. The third
step was to ligate the genes and linearized vector and transfer the ligation product into E. coli DH5α competent.
</div>
<div class="img-wrap no-margin">
<img class="w-40" src="https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering02.png" />
<span>Figure 2. The PCR gel electrophoresis picture of gene fragments.</span>
</div>
<div class="article-content">
Line M: DNA marker; Line 1: The gene fragment of gene crt ; Line 2: The gene fragment of gene hbd ; Line 3: The
gene fragment of gene ter; Line 4: The gene fragment of gene thlA.<br /><br />
We send the constructed recombinant plasmid to a sequencing company for sequencing, and the results proved that
the plasmid was successfully constructed (Figure 3). And the last step was extracting the recombinant plasmid from
E. coli DH5α and transferring it into Streptococcus Brevis ATCC367, so that can be used to produce N-butanol.
</div>
<div class="img-wrap no-margin">
<img class="w-60" src="https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering03.png" />
<span>Figure 3. The results of the sequencing data mapped to the plasmids</span>
</div>
<div class="title blue-title">How we produce N-butanol</div>
<div class="sub-title">a) Identification of transformed Streptococcus Brevis ATCC367</div>
<div class="article-content">
The constructed plasmid (containing 4 codon-optimized genes) was transformed into the Streptococcus Brevis ATCC367
by electroporation method and incubated on MRS solid medium plate at 37℃ for 24-48 hours. Next, we used colony PCR
to identify transformants (Figure 4). At this point, we got the engineered strain we wanted.
</div>
<div class="img-wrap no-margin">
<img class="w-40" src="https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering04.png" />
<span>Figure 4. Verification of transformant Streptococcus Brevis ATCC367</span>
</div>
<div class="article-content">Line M: DNA marker; Line 1: Plasmid positive control; Line 2-7: Different
transformants; Line 8: negative
control; Line9: blank control without any DNA template.</div>
<div class="sub-title">b) Measure the yield of N-butanol </div>
<div class="article-content">
The identified single colony was cultured in MRS overnight, and antibiotics were added and put into the anaerobic
chamber (this step can convert glucose into N-butanol) overnight. N-Butanol was detected by gas chromatography.
Finally, we analyzed all data using ultra-performance liquid chromatography, UPCL, and Q Exactive Orbitrap Mass
spectrometer to measure the yield of N-butanol (Figure 5). As shown in the result, we successfully detected a
higher yield of N-butanol. This result is really encouraging.
</div>
<div class="img-wrap no-margin">
<img class="w-40" src="https://static.igem.wiki/teams/4515/wiki/t-east-china-engineering05.jpg" />
<span>Figure 5. After pLY15-opt was transformed into Streptococcus Brevis, N-butanol production of ply15-opt
strain was measured at different times (48h, 69h, 95h, and 159h)</span>
</div>
<div class="title blue-title">How we learn from our projectHow we learn from our project</div>
<div class="article-content">
We have already collected the figures from our experiments. We developed an N-butanol-producing pathway in
Streptococcus Brevis ATCC367 and successfully increase the yield of N-butanol. With the development of society,
people pay more and more attention to the impact of environmental problems while improving their own lives.
Nowadays the greenhouse effect is serious, one of the important factors is the overuse of fossil energy.<br />
Because N-butanol is environmentally friendly energy, we believe that if we can use N-butanol in the future and
reduce our dependence on oil, it will relieve the pressure of resource shortage and make our environment better.
</div>
</div>
</div>
{% endblock %}
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