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− | As an incentive to participate in this Collaboration we gave away prizes. The five best teams that send us pictures got an E. Coli soft toy and the three team that sent us the most pictures got a bigger surprise. Thanks to Doulix, we provided these three Teams with customized trophies and vouchers.
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− | Team <a style="padding: 0;"href="https://2019.igem.org/Team:BOKU-Vienna"> BOKU-Vienna </a> was able to send us one picture, Team <a style="padding: 0;"href="https://2019.igem.org/Team:GO_Paris-Saclay"> GO Paris-Saclay</a> provided 3 pictures and Team <a style="padding: 0;"href="https://2019.igem.org/Team:TUDelft"> TUDelft</a> could help us with 14 colonie pictures. Team <a style="padding: 0;"href="https://2019.igem.org/Team:TU_Darmstadt"> TU Darmstadt </a> could send us a number of 23 pictures, but were beaten by Team <a style="padding: 0;"href="https://2019.igem.org/Team:IISER_Bhopal"> IISER Bhopal </a> which send us an impressive amount of 99 pictures.
| + | One mayor criteria to evaluate a chassis is its potential for an application project. To showcase these potential applications, our team decided to redirect the metabolic flux of Synechococcus elongatus UTEX 2973 in order to synthesis add-value compounds with C02 and light as a resource. As a target, we thought about a molecules, which also tackle one of the most important topic: the climate change. |
| </p> | | </p> |
− | <figure style="text-align: center; margin-right: 1em;"> | + | <br> |
− | <img src=https://static.igem.org/mediawiki/2019/0/04/T--Marburg--prizes_colony-picture.jpeg
| + | <p> |
− | alt="Prizes colony picture">
| + | Plains are considered to be one of the most environmental damaging transport devices commonly used (<a style="padding: 0" href=" https://pubs.acs.org/doi/full/10.1021/es9039693" target="_blank"> Borken-Kleefeld et al. 2010</a>). A quick estimation suggest, yet alone for the Giant Jamboree in Boston 2018, 14.000.000 CO2 was released for the flights. |
− | <figcaption>
| + | To counter this problem, we choose to set our focus on farnesene and limonene, which made 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 et al. 2015</a>). This fuel has proven to be suitable alternative to chemically made Oil based <a style="padding: 0" href=" |
− | Figure 1: Giving out the prizes for the best participants of the Colony-Picture Project at the giant Jamboree.
| + | 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> |
− | </figcaption>
| + | </p> |
− | </figure>
| + | <br> |
| + | <figure style="float:right; margin-left: 25px;"> |
| + | <img style="height: 500px; width: 750px;" |
| + | src=" https://2019.igem.org/File:T--Marburg--Metabolic--MEP-pathway.png" |
| + | alt="TeLuc and NanoLuc measurement in E.coli"> |
| + | <figcaption style="max-width: 550px; text-align: center"> |
| + | Fig. 1- Overvie of the MEP-Pathway. Enzymes are marked blue and Green. Limonene and Farnesen Synthase are summirsaed as TPS. Modified after Lin et al 2016 |
| + | </figcaption> |
| + | </figure> |
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| + | |
| + | <p> |
| + | Farnesene and limonene are both Terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). |
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| + | The MEP pathway is a conserved pathway in Bacteria and in photrophic organism, it uses C3-bodys deriving from the Calvin-Cycle (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodys, Isopentenyl pyrophosphate (IPP) and it´s Iso-mere Dimethylallyl pyrophosphate (DAMPP), are used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous express Limonene synthase to form D-Limonene, where FPP is a substrate for the plant derived Farnesene synthase. |
| + | Because Isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway in Cyanobacteria (<a style="padding: 0" href=" https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin et al. 2016</a>). |
| + | </p> |
| <br> | | <br> |
| <p> | | <p> |
− | We thank all the teams for sending us labeled colonie pictures. Without your help we would not have been able to train our AI and to let our opentrons OT-2 pick colonies. | + | As a the first step we decided to express heterologous the Limonen and the Farnesen Synthase to establish an overexpression strains. We used the Limonene synthase from … and the Farnesen synthase from … and codon optimised both enymes. |
| + | To redirect the flux into the MEP-Pathway we decided to overexpress the E.coli Proteins DXS, IDI and IspA. These targets were chosen on previous results, that could enhance the production of amorpha-4,11-diene to 19.8 mg/L in PCC 7942 without significant impairing the grwothrate (<a style="padding: 0" href=" https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-016-0617-8" target="_blank">Choi et al.2016</a>). |
| + | This surprisingly suggest, strong production capacities of production in Cyanobacteria. |
| </p> | | </p> |
− | </div>
| + | <br> |
− | </div> | + | <p> |
− | </div> | + | We chose the DXS, as many pathways are regulated on the first committing step. 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. |
| + | 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 in vitro (<a style="padding: 0" href=" http://www.jbc.org/content/282/4/2676.long" target="_blank">Xiang et al. 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 et al.2016</a>). |
| + | </p> |
| + | <br> |
| + | <p> |
| + | Removal of End product is a key component in overproduction strains. Because farnesene and limonene are volatile, the extra cellular diffusion rate is enough to prevent intracellular and poteinal toxic accumulation. On the other hand a nontoxic Overlay to catch the molecules is required, for example dodecan (<a style="padding: 0" href=" https://pubs.acs.org/doi/10.1021/acs.jafc.7b03625" target="_blank">Lee et al. 2017</a>). An alternative supply with CO2 is also required, therefore we copied the system from Lee et al and introduced a small tube with holes into the Medium. |
| + | </p> |
| + | <br> |
| + | <p> |
| + | |
| + | 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 et al. 2016</a>). |
| + | As IDI is an Isomerase, only a comparable small amount of Protein should be sufficient for enhanced effect, so Promotr <a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23110" target="_blank">BBa_J23110</a>. 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>. |
| + | |
| + | Sadly, we weren´t able to test our constructs in vivo and dropped this side project due to time reasons. As of now the submitted Parts for the limonene and the Farnesene synthase codon optimised for Synechococcus have been added to the registry. |
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| + | </p> |
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