Team:Marburg/test

G I A N T
J A M B O R E E


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.

Our team at the Giant Jamboree
Our team at the Giant Jamboree



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.




Afterwards, most of the time we had to stay with three or more people near our poster, 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.

Our team at the roof of the Opentrons building, during our visit in New York City
Our team at the roof of the Opentrons building, during our visit in New York City

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.

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!




Metabolic MEP Pathway
Fig. 1- Overview of the MEP-Pathway.
Enzymes are marked blue and Green.
Limonene and farnesene Synthase are summarised as TPS.
Modified after Lin et al 2016

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 as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s isomere dimethylallyl pyrophosphate (DAMPP) is used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous expressed limonene synthase to form D-Limonene, whereas 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 (Lin et al., 2016).

Our team at the roof of the Opentrons building, during our visit in New York City
Our team at the roof of the Opentrons building, during our visit in New York City

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.

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.

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Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra.

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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