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− | M E A S U R E M E N T | + | <h1 class="heading" style=" |
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| + | "> |
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| + | G I A N T<br> J A M B O R E E |
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| <section class="section"> | | <section class="section"> |
− | <h1 class="title">Metabolic Engeneering</h1>
| + | <div style="display: flex; flex-direction: row;"> |
− | <p style="margin-bottom: 1em;">
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− | One mayor criteria to evaluate a chassis is its potential for an application project. To showcase these potential applications, our team
| + | <p style="margin-bottom: 1em;"> |
− | decided to redirect the metabolic flux of Synechococcus elongatus UTEX 2973 in order to synthesize add-value compounds with CO<sub>2</sub> | + | Traditionally, we presented our project at the Giant Jamboree in Boston. It was a lot of fun to meet all the other iGEM teams and to talk and discuss with like-minded people. We loved watching the other team's presentations and listening to their detailed explanations at their posters. Thereby, we realised once again how broad the field of Synthetic Biology is and how big of an impact this field already is and how it will be even bigger in the future. With our presentation, poster and a booth at the Exhibition Space we showed what we were able to achieve in the last year. |
− | and light as a resource. As a target, we thought about molecules, which also tackle one of the most important topic: the climate change.
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| + | </p> |
| + | |
| + | <figure style="text-align: center; margin-left: 25px; max-width: 42000px"> |
| + | <img src="https://static.igem.org/mediawiki/2019/5/53/T--Marburg--GiantJamboree_2019_Grouppicture.jpg" |
| + | alt="Our team at the Giant Jamboree"> |
| + | <figcaption style="text-align: center;"> |
| + | Our team at the Giant Jamboree |
| + | </figcaption> |
| + | </figure> |
| + | |
| + | </div> |
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| + | <p style="margin-bottom: 1em;"> |
| + | <br> <br> Our presentation was one of the first on the first day. The room was so crowded, people were even standing in the doorway to watch our presentation! Here you have the possibility to see the presentation of our team at the Giant Jamboree. |
| + | </p> |
| + | <div style=" |
| + | position: relative; |
| + | height: 0; |
| + | width: 100%; |
| + | padding-bottom: 56.25%; |
| + | "> |
| + | <iframe src="https://www.youtube.com/embed/CwasIzE0R8o" frameborder="0" allow="accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture" allowfullscreen="" style="position: absolute;width: 100%;height: 100%;top: 0;left: 0;"></iframe> |
| + | </div> |
| + | |
| + | <br> |
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| + | <br> |
| + | <p style="margin-bottom: 1em;"> |
| + | <br> Afterwards, most of the time we had to stay with three or more people near our |
| + | <a style="padding: 0" href="https://static.igem.org/mediawiki/2019/2/2a/T--Marburg--Poster_2019_Marburg.pdf" target="_blank">poster</a>, because so many people were interested in our project. |
| + | Additionally, Keoni Gandall and Kristin Ellis paid us a visit at our Exhibition Space booth. They were really impressed by our custom-made hardware. |
| + | </p> |
| + | <figure style="float:right; margin-left: 25px; text-align: center;"> |
| + | <img style="height: 500px; mix-blend-mode: multiply;" src="https://static.igem.org/mediawiki/2019/9/93/T--Marburg--OpentronsVisitinNY_Grouppicture.jpg" |
| + | alt="Our team at the roof of the Opentrons building, during our visit in New York City"> |
| + | <figcaption style="text-align: center;"> |
| + | Our team at the roof of the Opentrons building, during our visit in New York City |
| + | </figcaption> |
| + | </figure> |
| + | <p style="margin-bottom: 1em;"> |
| + | Kristin was so happy about our work on the OT-2 Colony Picker Unit that she invited us to their company headquarters in New York City! When we went to Opentrons in New York City Kristin showed us their offices and labs, explained us how they work at Opentrons and we presented our Colony Picker Unit to several staff members, sharing our thoughts and ideas. |
| + | </p> |
| + | |
| + | |
| + | |
| + | <p style="margin-bottom: 1em;"> |
| + | Working independently and in collaborations on our own project, joining the Giant Jamboree together as a team and getting in touch with all these different, amazing people meant a lot to us and we are really thankful to have made these experiences! Finally we want to thank everybody who helped us gain a silver medal in this awesome competition! We are happy for this amazing time and to be a part of this great community! |
| </p> | | </p> |
− | <p style="margin-bottom: 1em;"> | + | |
− | Planes are considered to be one of the most environmentally damaging transport devices commonly used (<a style="padding: 0"
| + | <br><br><br> |
− | href=" https://pubs.acs.org/doi/full/10.1021/es9039693" target="_blank">Borken-Kleefeld<i> et al.</i>, 2010</a>). A quick estimation
| + | |
− | suggests, yet only for the Giant Jamboree in Boston 2018, 14.000.000 tons CO<sub>2</sub> was released from the flights.
| + | |
− | To encounter this problem, we chose to set our focus on farnesene and limonene, which make up 90 % of the biojetfuel AMJ700t (50% limonene,
| + | |
− | 40% farnesene) from Amyris (<a style="padding: 0" href=" https://aem.asm.org/content/aem/81/10/3316.full.pdf" target="_blank">Brennan<i> et
| + | |
− | al.</i>, 2015</a>). This fuel has proven to be a suitable alternative to chemically produced, oil based <a style="padding: 0"
| + | <figure style="float:right; margin-left: 25px; text-align: center;"> |
− | href="
| + | <img style="height: 500px; mix-blend-mode: multiply;" src="https://static.igem.org/mediawiki/2019/7/7d/T--Marburg--Metabolic--MEP-pathway.png" |
− | https://investors.amyris.com/2012-06-19-Photo-Release-Azul-Brazilian-Airlines-Makes-Successful-Demonstration-Flight-With-Amyris-Renewable-Jet-Fuel-Produced-From-Sugarcane" target="_blank">jet fuels</a>.
| + | |
− | </p>
| + | |
− | <figure style="float:right; margin-left: 25px;"> | + | |
− | <img style="height: 500px; width: 750px;" src="https://static.igem.org/mediawiki/2019/7/7d/T--Marburg--Metabolic--MEP-pathway.png" | + | |
| alt="Metabolic MEP Pathway"> | | alt="Metabolic MEP Pathway"> |
− | <figcaption style="max-width: 550px; text-align: center"> | + | <figcaption style="text-align: center;"> |
− | Fig. 1- Overview of the MEP-Pathway. Enzymes are marked blue and Green. Limonene and farnesene Synthase are summarised as TPS. Modified | + | Fig. 1- Overview of the MEP-Pathway.<br> |
− | after Lin et al 2016 | + | Enzymes are marked blue and Green.<br> |
| + | Limonene and farnesene Synthase are summarised as TPS.<br> |
| + | Modified after Lin et al 2016 |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
| <p style="margin-bottom: 1em;"> | | <p style="margin-bottom: 1em;"> |
| Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). | | Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). |
− |
| |
| The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle (directly | | The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle (directly |
| as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s | | as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s |
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| 2016</a>). | | 2016</a>). |
| </p> | | </p> |
− | <p style="margin-bottom: 1em;">
| + | |
− | As a the first step we decided to heterologous express the limonene and the farnesene synthase to establish overexpression strains. We used | + | |
− | the limonene synthase from <i>Lavandula angustifolia</i> and the farnesene synthase from <i>Actinidia deliciosa</i> and codon optimised both
| + | |
− | enzymes.
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− | To redirect the flux into the MEP-Pathway we decided to overexpress the <i>E.Coli</i> proteins DXS (1-deoxy-D-xylulose-5-phosphate
| + | |
− | synthase), IDI (Isopentenyl-diphosphate Delta-isomerase) and IspA (Farnesyl diphosphate synthase). These targets were chosen on based
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− | previous results, which showed enhancement the production of amorpha-4,11-diene to 19.8 mg/L in PCC 7942 without significant impairing the
| + | |
− | growth rate (<a style="padding: 0" href=" https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-016-0617-8"
| + | |
− | target="_blank">Choi<i> et al.</i>,2016</a>). | + | |
− | Surprisingly, this suggest strong capacities of production in Cyanobacteria and no build-up of toxic intermediates.
| + | |
− | </p> | + | |
| + | |
| + | |
| + | <figure style="float:right; margin-left: 25px; text-align: center;"> |
| + | <img style="height: 500px; mix-blend-mode: multiply;" src="https://static.igem.org/mediawiki/2019/9/93/T--Marburg--OpentronsVisitinNY_Grouppicture.jpg" |
| + | alt="Our team at the roof of the Opentrons building, during our visit in New York City"> |
| + | <figcaption style="text-align: center;"> |
| + | Our team at the roof of the Opentrons building, during our visit in New York City |
| + | </figcaption> |
| + | </figure> |
| <p style="margin-bottom: 1em;"> | | <p style="margin-bottom: 1em;"> |
− | Many pathways, such as the MEP-Pathway, are regulated on the first committing step. Therefore we chose the DXS as a first target.
| + | |
− | The IDI, would convert DMAPP to IPP, which is necessary to balance both pools, especially interesting for overproduction of Farnesene, where
| + | |
− | a ratio of 1:2 in favour of IPP is required.
| + | |
− | </p>
| + | |
− | <p style="margin-bottom: 1em;">
| + | |
− | IspA can synthesise GPP and IPP to FPP, which would improve the efficiency in a farnesene production strain.
| + | |
− | To further enhance the efficiency of this pathway, we decided to mutate the DXS-residue 392 from a thyrosine (Y) to a phenylalanine (F).
| + | |
− | This would lead to a threefold increase in activity <i>in vitro</i> (<a style="padding: 0"
| + | |
− | href=" http://www.jbc.org/content/282/4/2676.long" target="_blank">Xiang<i> et al.</i>, 2016</a>).
| + | |
− | Also it would be advisable to remove the allosteric feedback regulation of the DXS (<a style="padding: 0"
| + | |
− | href=" https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161534” target=" _blank">Banerjee<i> et al.</i>,2016</a>).
| + | |
− | </p>
| + | |
| <p style="margin-bottom: 1em;"> | | <p style="margin-bottom: 1em;"> |
− | Removal of the end product is a key component in overproduction strains. Because farnesene and limonene are volatile, the extracellular
| + | Traditionally, we presented our project at the Giant Jamboree in Boston. It was a lot of fun to meet all the other iGEM teams and to talk and discuss with like-minded people. With our presentation, poster and a booth at the Exhibition Space we showed what we were able to achieve in the last year. |
− | diffusion rate is enough to prevent intracellular and potential toxic accumulation. On the other hand, a nontoxic overlay to catch the
| + | |
− | molecules is required, for example dodecan can be used. (<a style="padding: 0" href=" https://pubs.acs.org/doi/10.1021/acs.jafc.7b03625"
| + | </p> |
− | target="_blank">Lee<i> et al.</i>, 2017</a>). An alternative supply with CO2 is also required, therefore we copied the system from Lee
| + | |
− | <i>et al. </i> and introduced a small tube with holes into the Medium.
| + | |
− | </p>
| + | |
− | <p style="margin-bottom: 1em;">
| + | |
− | To balance the pathway we decided to use a weak promotor (<a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23103"
| + | |
− | target="_blank">BBa_J23103</a>) for the ispA, as competition over FPP could lead to a heavy growth impact (<a style="padding: 0"
| + | |
− | href=" https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin<i> et al.</i>, 2016</a>).
| + | |
− | As IDI is an Isomerase, only a comparably medium amount of protein should be sufficient for a enhanced effect, so Promotor <a
| + | |
− | style="padding: 0" href="http://parts.igem.org/Part:BBa_J23110" target="_blank">BBa_J23110</a> was chosen. Because the DXS is the a
| + | |
− | potential bottle neck in Terpene Production, we chose the Promotor <a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23111"
| + | |
− | target="_blank">BBa_J23111</a>.
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | Sadly, we weren´t able to test our constructs <i>in vivo</i> and dropped this side project due to time reasons. As of now the submitted
| + | |
− | Parts for the limonene and the Farnesene synthase, which have been codon optimised for UTEX 2973 have been added to the registry ( <a
| + | |
− | style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228051" target="_blank"> BBa_K3228051</a> and <a style="padding: 0"
| + | |
− | href=" http://parts.igem.org/Part:BBa_K3228052" target="_blank"> BBa_K3228052</a>).
| + | |
− | </p>
| + | |
| </section> | | </section> |
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