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Commit c7326872 authored by Sasha Fujiwara's avatar Sasha Fujiwara
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{% endblock %}
{% block lead %}Make a useful contribution for future iGEM teams. Use this page to document that contribution.{% endblock %}
{% block lead %}Make a useful contribution for future iGEM teams. Use this page to document that contribution.{%
endblock %}
{% block page_content %}
<!-- Main Content -->
<div class="row">
<div class="col-2"></div>
<div class="col-8 ps-5">
<h1>Overview</h1>
<p>Throughout our exploration of PHB production and secretion system, our team was able to make a number of
contributions to the field of bioplastic production as well as the growing realm of synthetic biology. The
following
lists our most significant contributions from this our project ESPHA: an extented collectivized literature
review
regarding PHA bioplastics as a whole and PHA synthesis mechanisms, a model for PHB accumulation for
production
scaling, new parts for generalized and PHB-specific secretion models, and an early characterization of
vesicle
nucleating peptides (VNps). </p>
</div>
</div>
<!-- Side Bar -->
<div class="col-md-4 d-none d-md-flex justify-content-center" style="padding: 5vh 0">
{% include 'sidebar.html' %}
</div>
<!-- Main Content -->
<div class="col-md-6 col-10 order-2">
<div class="row mt-4">
<div class="col">
<div class="bd-callout bd-callout-info">
<h4>Bronze Medal Criterion #4</h4>
<p>Make a useful contribution for future iGEM teams. Use this page to document that contribution.
<p>
<p>If you are making a contribution by adding information to an existing Part or creating a new Part,
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. You can use this page to link to that part and include additional information about your
contribution.</p>
<hr>
<p>Please see the <a href="https://competition.igem.org/judging/medals">2023 Medals Page</a> for more
information.</p>
</div>
</div>
<div class="row mt-4 sideline">
<h1 id="ss-PHA">PHA</h1>
<h3>Extended Literature Review</h3>
<p>Throughout the progression of our project, we often found ourselves immersing in various literature reviews
and analyzing numerous data from past iGEM teams and field-specific research. Due to the large complexity of
PHA production, we have collectivized all of our references and resources into a single extended literature
review. We hope future teams as well as research groups may find our organized information helpful for their
projects and explorations. </p>
<h3>Our Results</h3>
<p>In Project ESPHA, we have demonstrated the applied integration of PHB production with effective secretion
methods. We have analyzed and compared results from a number of secretion systems, including the type I Hlya
secretion system, type II TorA secretion system, and VNps. Our gathered results may be used in future models
of PHB producing systems in E. Coli. Further, our research explores the potential for reducing downstream
production costs of PHB and other types of PHA production models via the modification of the extraction
stage, paving a path towards a future of PHA production that is less costly, environmentally friendly, and
scalable. </p>
<h3>Model for Scaling</h3>
<p>One of the key focuses of project ESPHA has been scalability. While our research delved into the specific
mechanisms of PHB secretion, our larger context for our project is to demonstrate the potential for PHB and
other bioplastics to compete in the plastic market. In doing so one of the most important aspects of PHB
production is the cost to yield ratio for largescale models. Through <a
href="https://2023.igem.wiki/asij-tokyo/human-practices">Human Practices</a>, we have reached out to
several stakeholders in the bioplastic and plastic industries for their input and perspectives into the
current field. However, in addition to that, we have developed a model for comparing PHA accumulation with
respect to the change in carbon and nitrogen levels. In other words, our model examines how fast carbon or
nitrogen levels in cell cultures diminishes according to differing PHA accumulation rates. Therefore, the
model can be used to as a framework for largescale PHA production involving carbon and nitrogen-based
resources for industrial microbial cultures. Our constructed model can be found on the <a
href="https://2023.igem.wiki/asij-tokyo/modeling">Modeling page</a>. </p>
</div>
<div class="row mt-4 sideline">
<h1 id="ss-SecretionSystems">Secretion Systems</h1>
<h3>Parts</h3>
<p>Adding on to the growing collection of BioBrick parts in the registry, we have contributed a total of 3 new
basic parts and 21 new composite parts this year. While we have utilized most of the secretion tags for
secreting PHB for the purposes of our project, these secretion tags and systems could be modified to
accommodate a variety of other uses including, but not limited to, alternative proteins, and drug delivery
systems. Additional documentation of our used and designed parts can be found on the <a
href="https://2023.igem.wiki/asij-tokyo/parts">Parts
page</a>. </p>
<h3>Our Results</h3>
<p>In Project ESPHA, we have demonstrated the applied integration of PHB production with effective secretion
methods. We have analyzed and compared results from a number of secretion systems, including the type I Hlya
secretion system, type II TorA secretion system, and VNps. Our gathered results may be used in future models
of PHB producing systems in E. Coli. Further, our research explores the potential for reducing downstream
production costs of PHB and other types of PHA production models via the modification of the extraction
stage, paving a path towards a future of PHA production that is less costly, environmentally friendly, and
scalable. </p>
<h3>Vesicle Nucleating Peptides (VNps)</h3>
<p>One of our most promising contributions this year was our use and documentation of vesicle nucleating
peptides (VNps). Our team discovered the potential use of VNps after coming across an article published by
Eastwood et. al describing a novel system in which proteins–such as those that are typically challenging to
produce in E. Coli–could be exported through membrane-bound vesicles with a single peptide tag called VNps.
Not only are VNps a viable alternative, they provide further advantages to traditional secretion systems as
well. As previously implied, VNps allow for the isolated production and storage of proteins with insoluble,
toxic properties. Vesicles also provide a stable, long-term environment for proteins. VNps have been proven
to possess numerous flexible characteristics. Eastwood et. al, originally explored the possibility of
improving the tag itself by making simple modifications to the VNp amino acid sequence. Their results found
that they could enhance protein yields, enhance vesicular export over a wider range of culture temperatures,
and shorten the sequence to just 20 residues while working in a variety of E. Coli strains. Further, VNps
are compatible with a variety of plasmids, promoters, and induction levels. Moreover, the vesicle system can
be used for the expression of a wide range of recombinant proteins.
<br><br>
Through our project, we have successfully characterized many of these features. Firstly, we have confirmed
the functionality of VNps in our BL21 DE3 E. Coli strain. In addition, we have demonstrated the
implementation of VNps in a practical application of PHB secretion via a VNp-Phasin-GFP fusion protein
design. While we utilized the VNp6 variant in particular for our PHB secretion model, we have provided parts
designs for two other promising variants, VNp2 and VNp15. We have added all three VNp basic parts designs to
the registry while acknowledging the fact that the parts were uploaded to the Parts Registry by ASIJ-Tokyo
iGEM but have been designed by an outside lab. We hope future teams and research may apply similar if not
innovative methods for utilizing vesicle nucleating peptides to their designs.
</p>
</div>
</div>
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