Difference between revisions of "Team:Marburg/test"

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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.
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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 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.  
 
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                    <img src=https://static.igem.org/mediawiki/2019/0/04/T--Marburg--prizes_colony-picture.jpeg
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                      alt="Prizes colony picture">
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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.
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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="
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                      Figure 1: Giving out the prizes for the best participants of the Colony-Picture Project at the giant Jamboree.
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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>
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              src=" https://2019.igem.org/File:T--Marburg--Metabolic--MEP-pathway.png"
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              alt="TeLuc and NanoLuc measurement in E.coli">
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              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
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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.
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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>).
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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.
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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.
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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>).
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This surprisingly suggest, strong production capacities of production in Cyanobacteria.  
 
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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.
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IspA can synthesise GPP and IPP to FPP, which would improve the efficiency in a Farnesene production strain.
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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>).
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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>).
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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.
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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>).
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  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>.
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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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Revision as of 18:10, 8 December 2019

M E A S U R E M E N T

Amplifying new standards in measurement

Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS)

Storytelling:

We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX 2973, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us in front of many problems and questions. Especially the usage of different media, light conditions and other cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers. Many of these problems are reasoned in the ongoing optimization and development of methods and instruments. Therefore it is hard to hold on to special methods but still standardization is a huge part in synthetic microbiology and necessary to compare results with other scientists and reproduce their data.

While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in light measurement, evaluated different reporters???, established a measurement method and compared it to a already known FACS measurement method (?).

At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle]. So we had to measure the light conditions in our incubators and while doing this simple task the first part of standardization began. We discovered that nearly every paper? is using different methods to measure their light conditions and that it is a really complex and important procedure. So we got in contact with cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following popup we show different ways of measurement, their (dis-)advantages and different results depending on the measuring instrument.
Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. In literature and while talking with different experts (IHP), we recognized that small deviations of these parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters that lead to the fastest growth speed.
Another aspect was measuring the expression and characterize our part. Different possibilities were discussed and after testing them we decided on two methods in our project (plate reader and FACs). One approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.
The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the difference in measurement methods.
At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973 and make a contribution to the cyano community by establishing essential/fixed standards in measurement.

L I G H T
M E A S U R E M E N T

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R E P O R T E R S

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F A C S

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P A R T
M E A S U R E M E N T

For our project it was indispensable to establish a measurement workflow that is not only applicable to UTEX 2973 and other cyanobacteria but also has a high throughput.

G R O W T H
C U R V E S

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