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        C O L L A B O R A T I O N S
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            I M P R O V E
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      </h1>
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            <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" class="logo" alt="Syntex Logo">
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        <h1 class="title"> </h1>
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            <h1 class="title"></h1>
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        <p style="text-align: justify;">
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            <p style="text-align: justify;">
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          Through this iGEM year we interacted with so many iGEM Teams all around the world. It was a pleasure working
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                Every Synbio Experiment is more or less based on the same principle: You change a system in some way and
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          with all the different teams and learn from each other. We got so many impressions about other projects and
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                you look at the outcome. This readout is one of the most important things in all natural science, a
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          hope we could help with our collaborations. A huge Thank you for every team collaboration!
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                wrong readout can easily flaw your whole experiment or can lead to serious misconclusion.<br>
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        </p>
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                The most common way to measure localisation, interaction or even the intensity of genetic elements is
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      </section>
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                via Fluorescence as readout.<br>
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      <hr>
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                Fluorescence Proteins (FP), started with the green fluorescent protein, are based on the ability of a
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      <section class="section grid">
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                chromophore to absorb photons of specific wavelength and emit this photon at another. Even on the iGEM
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                registry, the characterization via FPs is the suggested way to characterise a part.<br>
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          <div class="sub-header">
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                This Method is prone to Background noise, depends on the folding of the Protein at the specific cell
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            <h1>
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                conditions and furthermore the chromophore can even bleach after to much exposure, so the drawbacks are
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              C O L O N Y<br>
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                obvious.<br>
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              P I C T U R E S
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                <br>
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            </h1>
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                Bioluminescence could make the desired difference, but the original Luciferase Assays either consistent
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            <hr>
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                of an whole Operon systems, or put an unnecessary high metabolic burden through ATP dependency and/or
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                trough its relatively large size (Firefly-Luciferase 61,5 kDa). Together with the low quantity, which
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          <div class="sub-content">
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                can be several orders of magnitude lower than a fluorescence based system, the common breakthrough of
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            <p>
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                Lumincese in Synthetic biology is still missing.<br>
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              In our colony picking collaboration we asked other iGEM teams to supply us with data for our picking
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                Newly developed small ATP independent Lucferase Proteins, are interesting candidates to bypass these
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              algorithm. Thanks to this group effort we could build our colony picking unit.
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                Problems. Nanoluc, with its 19 kDa and up to 150 fold increase in brightness compared to the
 +
                Firefly-Luciferase is handled as an suitable alternative. This Protein use the patented Substrate
 +
                Furimazine, and emits Photons at 460 nm. Naoluc has been successfully implemented in Promoter testing
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                and as an alternative in Interaction messurement via Bilumiecnce Resonace energy transfer, but sadly
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                only few team ever used this system.<br>
 +
                One scratch on the surface of Nanoluc is for sure the restriction of the wavelength. While for
 +
                Measurements in many organisms and Tissues, this looming Problem did not occur, it's becoming obvious,
 +
                when looking into phototrophic Organisms and deep-tissue mammalian cells. As the keen reader might
 +
                guess, cells absorb Light of the wavelength under 600 nm to a great extent and even more if they have a
 +
                photosystem. Chlorophyll a have one their two peaks at 440 nm [fig.1]. If one would compare that with
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                the nanoluc spectra, a devastating conclusion could be made: The Photosystem will absorb photons from
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                the Signal, leading to weaker peaks, and maybe more grave/frightening/alarming, a dependency of Signal
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                on the chlorophyll content. Althroug localisation experiments should´t be affected that much,
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                Measurement and characterisation, the foundation of which synthetic Biology is build on, could be
 +
                shaken.
 +
                Driven by this problem, we dig ourselves in literature and found a solution: A mutated Version of
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                nanoLuc, so called teLuc<br>
 +
                <a href="http://parts.igem.org/Part:BBa_BBa_K3228042_BBa_K3228042"> BBa_BBa K3228042 BBa K3228042 </a>
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                <br>
 +
                which has a severe red shifted pattern with a peak at 502 nm (Figure 2). What is even more serviere is
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                the astonishing brightness, wich even surpass nanoluc by several folds (5,7) in vitro. In vivo this
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                effect is even more dramatic, through its ability to bypass the absorption of Light. We expect this
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                ability of teLuc to surpass the limits of Luminescence in plants to an amazing extent, and allow the
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                plant synthetic biology community to accelerate their research.<br>
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                <figure style="text-align:center">
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                        <img style="height: 250px; width: 900px;"
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                            src="chrome.google.com/webstore/category/collection/chrome_themes?hl=de
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                  " alt="Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra">
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                        <figcaption style="max-width: 2400px; text-align: center">
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                            Fig.2 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra
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                        </figcaption>
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                    </figure>
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                <br>
 
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                     src="https://lh3.googleusercontent.com/8ko4suiu3NQ_qmRIeZf1k1sg5EUw8g4JXfkGG3xAmRk1dxaVlZQbzC9Uz-6ToGKXaAf5p_yx9MVHhlO3QdMmG_l0ukJ0OVQOWBzcouM-HOTc_ta7LblxiVtTdLKrf9q4bpzP6ZRP"
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                    alt="Lvl1 ori">
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                <figcaption>
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              <h1 class="title">Colony Pictures</h1>
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                     Fig.1 - Lvl1 ori
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                 <h2 class="subtitle">The Colony picture Collaboration</h2>
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                  <figure style="margin-right: 25px;">
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                    <img src=https://static.igem.org/mediawiki/2019/5/5e/T--marburg--colony_picking_2.jpeg
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                      alt="Bild von Roboter Platzhalter">
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                    <figcaption>
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                      Figure 1: Pipetting Robot (OT-2) colony picking set up.
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                    </figcaption>
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                      Many repetitive tasks in the lab cost time such as pipetting or picking colonies from agar plates
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                      and could technically be performed by robots. We used Opentrons OT-2 as a platform and designed
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                      hardware modules and scripts to outsource these tasks. Thereby, we can now offer the Opentrons
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                      user community an automated pipeline to streamline laboratory practices in ways that haven’t been
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                      possible before.
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                    </p>
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                    <p>
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                      For the software we decided to use an artificial intelligence (AI). Artificial intelligences
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                      however rely on huge data sets to learn their specific task to perfection. In our case, we chose
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                      pictures of agar plates with colonies of E. coli as a suitable training set. Our experiments
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                      resulted in a big amount of agar plates but it was clear to us pretty quickly that we could never
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                      reach a big enough data set just by making photos of our own plates to get an AI with an adequate
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                      performance. To achieve this goal, we launched the Colony picture collaboration. We asked other
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                      iGEM Teams if they could provide us pictures of their agar plates with E. coli colonies with which
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                      we can then train the robot. In order to do that we published a script on how to take the ictures.
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                      We made the script as precise as possible to make sure that the data set was as consistent as
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                      possible.
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                    Following this, we built a website so they could label the colonies on their images online.
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                  </p>
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                  <figure style="margin-left: 25px;">
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                     <img src="https://static.igem.org/mediawiki/2019/f/f8/T--marburg--taking_colony_pictures.png"
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                      alt="Pattern for taking the colony picture">
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                    <figcaption>
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                      Figure 2: Pattern for taking the colony picture.
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                     </figcaption>
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                  </figure>
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                  <figure style="text-align: center; margin-right: 25px;">
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                    <img src="https://static.igem.org/mediawiki/2019/0/0b/T--marburg--collonycollab_website.png"
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                      alt="colony picking website">
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                    <figcaption>
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                      Figure 3: Website for colony picutre labeling.
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                    </figcaption>
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                  </figure>
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                  <p>
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                    In total 250 colony pictures were added to our data set, vastly increasing the performance of our AI
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                    in detecting colonies. As an incentive to participate in this collaboration we gave away prizes to
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                    the teams that sent us their pictures, they received a small surprise, an E. coli plushie. The three
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                    teams that sent us the most pictures got an engraved trophy out of glass thanks to our sponsor of
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                    this collaboration, Doulix.
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                  </p>
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            <h1>T H E<span style="visibility: hidden;">A</span>G O L D E N<span style="visibility: hidden;">A</span>G A
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              T E</h1>
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            <h1>C O L L A B O R A T I O N</h1>
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            <hr>
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          </div>
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          <div class="sub-content">
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            <p>
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              Our team made itself the goal to introduce other iGEM Teams to Golden Gate. Therefore we hosted a webinar, build up a
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              Communication Platform and made an Interlab.
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            </p>
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              <h1 class="title">The Golden Gate collaboration</h1>
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                 <p>
 
                 <p>
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                  Earlier this year the iGEM headquarters announced changes in their part submission system, one of
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                    Introduction of exogenous DNA can be done in multiple ways and propagated in a strain if it is
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                  which made us extremely happy: BioBricks are now also accepted in Type IIS standard! In our opinion,
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                    integrated in the
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                  this is a huge step, as cloning techniques based on Type IIS restriction enzymes are becoming more
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                    chromosome or stably expressed on a self-replicating plasmid.<br> For rapid prototyping in
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                  and more popular in the scientific community due to their numerous advantages over classical cloning
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                    cyanobacteria
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                  methods.<br>
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                    self-replicating plasmids are of higher interest than genome-integrations, as the latter can be
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                  We noticed that more teams converted to MoClo systems in their projects this year but we still think
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                    quite
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                  there is a lot that needs to be done for the iGEM community to really embrace this change. In order
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                    time-consuming in cyanobacterial strains with multiple genome copies (<a
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                  to help teams with the transition into this new system, we had an idea:
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                        href="https://www.ncbi.nlm.nih.gov/pubmed/22092711">Griese <i>et al.,</i> 2011</a>).
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                  What if we could help other teams understand what Type IIS enzymes really are and how they work? If
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                    Furthermore, genes
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                  we could share our passion for Modular Cloning and the value of the PhytoBrick standard? If we could
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                    introduced in self-replicating vectors have been shown to have higher gene-expression levels than
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                  introduce them to our Marburg Collection and help removing some of the burdens that come with a
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                    those integrated
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                  switch to a new cloning system?<br>
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                    in the genome, as copy numbers are typically higher (<a
−
                  Therefore we hosted a webinar about Golden Gate, build up a communication platform to give iGEM
+
                        href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt
−
                  Teams a chance to troubleshoot their methods and made an Interlab study. Here we evaluated the
+
                        übernehmen <i>et
−
                  consistency of Golden Gate Assembly protocols across iGEM teams as well as to give teams the
+
                            al.,</i> 2016</a>) – a desirable trait, not just for rapid prototyping in research
−
                  opportunity to gain experience with the assembly method.
+
                    applications, but also for
 +
                    biotechnological production of valuable compounds.<br>
 +
                    With our shuttle-vectors encompass a cyanobacterial origin of replication (ori) from
 +
                    <i>Synechococcus
 +
                        elongatus</i> PCC7942 as well as an <i>E.coli</i> ori, which is perfect for fast cloning
 +
                    processes, as these
 +
                    vectors can be easily recovered from the cyanobacteria and reintroduced in an <i>E.coli</i>
 +
                    strain.<br>
 +
                    <br>
 +
                </p>
 +
                <br>
 +
                <p style="font-size: 20px">
 +
                    Currently existing shuttle vectors for cyanobacteria are still based on standard systems working
 +
                    with multiple
 +
                    cloning sites (MCS) for expression of homologous genes (<a
 +
                        href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt
 +
                        übernehmen <i>et
 +
                            al.,</i> 2016</a>). A huge downside is that these vectors include either an MCS (e.g.
 +
                    pAM5188) or a
 +
                    fluorescence reporter (e.g. pAM4787), which is unpractical for easy selection of recombinant clones.
 +
                    Additionally,
 +
                    an MCS comes with possible sequence constraints due to restriction sites leaving unwanted base pairs
 +
                    in your
 +
                    constructs.<br>
 +
                    Facilitating and standardizing the process of engineering biological systems is one of the
 +
                    fundamental goals of
 +
                    synthetic biology (<a href="https://doi.org/10.1186/1754-1611-2-5">Shetty Titel anhand dieser DOI in
 +
                        Citavi-Projekt übernehmen <i>et al.,</i> 2008</a>), so the
 +
                    construction of a shuttle-vector based on a modular cloning method significantly improves the
 +
                    genetic toolbox we
 +
                    created for genetic engineering and synthetic biology approaches in <i>S.elongatus</i> and other
 +
                    cyanobacteria.<br>
 +
                </p>
 +
                <br>
 +
                <p style="font-size: 20px">
 +
                    The commonly used <i>S.elongatus</i> strain PCC7942 carries two endogenous plasmids, the 46,4kb pANL
 +
                    (<a href="https://www.ncbi.nlm.nih.gov/pubmed/18353436">Chen <i>et al.,</i> 2008</a>) which is
 +
                    essential and the
 +
                    7,8kb pANS (<a href="https://www.ncbi.nlm.nih.gov/pubmed/1552863">Van der Plas <i>et al.,</i>
 +
                        1992</a>) which is
 +
                    not essential for the strain and can easily be cured.<br>
 +
                    This small plasmid has already been used for construction of shuttle vectors (<a
 +
                        href="https://doi.org/10.1016/0076-6879(87)53054-3">Kuhlemeier Titel anhand dieser DOI in
 +
                        Citavi-Projekt übernehmen & van Arkel, 1987</a> ; <a
 +
                        href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC217787/">Golden & Sherman, 1983</a> ; <a
 +
                        href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt
 +
                        übernehmen <i>et al.,</i> 2016</a>). <br>
 +
                    We followed this lead to create the best shuttle-vector available for cyanobacteria by encompassing
 +
                    the minimal
 +
                    replication region of pANS and the ColE1 origin of replication into our vectors, allowing for stable
 +
                    self-replication with high copy numbers in cyanobacteria (<a
 +
                        href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt
 +
                        übernehmen
 +
                        <i>et al.,</i> 2016</a>) and <i>E.coli</i> (<a
 +
                        href="https://doi.org/10.1016/S0065-2660(02)46013-0">Gerhart Titel anhand dieser DOI in
 +
                        Citavi-Projekt übernehmen
 +
                        <i>et al.,</i>2002</a>). This addition to the genetic toolbox proves invaluable, as it can be
 +
                    easily recovered
 +
                    from the cyanobacterial strain and reintroduced in <i>E.coli</i> for fast GoldenGate-based cloning
 +
                    processes.<br>
 +
                </p>
 +
                <br>
 +
                <p style="font-size: 20px">
 +
                    In order to supply the community with an easy selection system, we equipped our shuttle vector with
 +
                    a fluorescent
 +
                    reporter that is cut out when introducing new genetic parts:<br>
 +
                    A mRFP (red fluorescent protein) cassette is flanked by our standardized TypeIIS restriction enzyme
 +
                    recognition
 +
                    sequences (BsmBI or BsaI depending on what level you want to clone in). In a standard Golden Gate
 +
                    reaction this
 +
                    cassette will drop out and leave space for the parts that should be introduced, allowing for easy
 +
                    selection on
 +
                    plate after successful cloning – red colonies are wrong, still harboring the mRFP cassette and white
 +
                    colonies (if
 +
                    no other fluorescence is introduced) are correct, as the mRFP was switched with the parts of
 +
                    interest.<br>
 +
                    <br>
 +
                    This crucial part comes in two variations - one for cloning Lvl1 and one for Lvl2 constructs -,
 +
                    giving the Golden
 +
                    Gate community everything they need for successful and reliable creation of self-replicating vectors
 +
                    in
 +
                    cyanobacteria.
 +
                </p>
 +
                <br>
 
                 </p>
 
                 </p>
−
              </section>
 
−
              <section class="section">
 
−
                <div class="wrap-collabsible" style="margin-bottom: 25px;">
 
−
                  <input id="collapsible2_1" class="toggle" type="checkbox">
 
−
                  <label for="collapsible2_1" class="lbl-toggle">
 
−
                    Webinar - Educating about Golden Gate cloning
 
−
                    <hr style="width: unset;">
 
−
                  </label>
 
−
                  <div class="collapsible-content">
 
−
                    <div class="content-inner" style="text-align: justify; text-align-last: left;">
 
−
                      <div style="display: flex; flex-direction: row;">
 
−
                        <figure style="text-align: center; margin-right: 25px;">
 
−
                          <img src="https://static.igem.org/mediawiki/2019/0/01/T--marburg--Webinar_button.png"
 
−
                            alt="Webinarbutton">
 
−
                        </figure>
 
−
                        <p>
 
−
                          We wanted to reach as many teams as possible with our webinar on Golden Gate cloning,
 
−
                          Type IIS enzymes, their benefits and how to use all of this in their own projects. We
 
−
                          started planning everything that we thought should be in the webinar, from the
 
−
                          biological function of the Type IIS enzymes and how they differ from Type II enzymes,
 
−
                          over its applications in Synthetic Biology like modular cloning to the in-depth
 
−
                          strategies when designing your own parts and actually building them in the lab.<br>
 
−
                          During this planning step we realized, that some of the teams that are already working
 
−
                          with Golden Gate might not benefit as much from an entry level webinar. Consequently, we
 
−
                          thought about integrating more complex design questions that we could share with the
 
−
                          community. We finished our webinars with a practical part, showing the part-design in
 
−
                          Geneious Prime – the sequence analysis software we used throughout the year. Firstly, we
 
−
                          explained the different levels that can be built with Golden Gate and then introduced
 
−
                          some more complex designs that are also possible with Golden Gate cloning, such as the
 
−
                          construction of whole operons or space holder parts. Through this we hoped to also reach
 
−
                          teams that use Golden Gate themselves, showing them the possibilities this cloning
 
−
                          system offers.<br>
 
−
                          To reach as many teams on the globe as possible we set up webinars on three different
 
−
                          times to cover as many time zones within a comfortable time frame – hoping that this
 
−
                          will allow more iGEM teams to participate.
 
−
                          The one and a half hour webinars were held on July 30th 10am CET, July 31st 6pm CET and
 
−
                          the last time on 8th August 12pm CET by our instructor René Inckemann with the support
 
−
                          of three of our team members.
 
−
                          We were happy that in total 18 teams participated, as well as some students from the
 
−
                          universities of Marburg and Gießen and even a few fellow scientists from the
 
−
                          Max-Planck-Institute for terrestrial microbiology. In total we counted around 70
 
−
                          participants. We were also really happy to see Sota Hirano from Doulix on our webinar,
 
−
                          who gave us some awesome feedback, stating that he did learn new things about Golden
 
−
                          Gate which he was not aware of before!
 
−
                        </p>
 
−
                      </div>
 
−
                      <div style="display: flex; flex-direction: row; margin-top: 25px">
 
−
                        <div>
 
−
                          <p>
 
−
                            If you would like to learn about Golden Gate assembly please feel free to watch our
 
−
                            webinar through this Link:
 
−
                          </p>
 
−
                          <div class="content">
 
−
                            <ul>
 
−
                              <li><a href="https://www.youtube.com/watch?v=-UUUG2iL7og">Webinar 30.07.2019</a></li>
 
−
                              <li><a href="https://www.youtube.com/watch?v=Aq-6MlaVWww">Webinar 31.07.2019</a></li>
 
−
                              <li><a href="https://www.youtube.com/watch?v=Aq-6MlaVWww">Webinar 09.08.2019</a></li>
 
−
                            </ul>
 
−
                          </div>
 
−
                          We thank all the following teams for participating in this awesome event:
 
−
                          <div class="content">
 
−
                            <ul>
 
−
                              <li><a href="https://2019.igem.org/Team:Aachen/collaborations">Team Aachen</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:Athens/Collaborations">Team Athens</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:Amazonas-Brazil/Collaborations">Team
 
−
                                  Amazonas-Brazil</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:CMUQ/Collaborations">Team CMU-Q</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:ITESO_Guadalajara/Collaborations">Team ITESO</a>
 
−
                              </li>
 
−
                              <li><a href="https://2019.igem.org/Team:SoundBio//Collaborations">Team Soundbio</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:UFPR-Curitiba-Brazil/Collaborations">Team
 
−
                                  UFPR-Curitiba Brazil</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:TUDelft/Collaborations">Team Delft</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:BOKU-Vienna/Collaborations">Team BOKU-Viena</a>
 
−
                              </li>
 
−
                              <li><a href="https://2019.igem.org/Team:Duesseldorf/Collaborations">Team Düsseldorf</a>
 
−
                              </li>
 
−
                              <li><a href="https://2019.igem.org/Team:IISc-Bangalore/Collaborations">Team
 
−
                                  IlSc-BAngalore</a>
 
−
                              </li>
 
−
                              <li><a href="https://2019.igem.org/Team:TU_Kaiserslautern/Collaborations">Team
 
−
                                  Kaiserslautern</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:Leiden/Collaborations">Team Leiden</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:UM_Macau/Collaborations">Team Macau</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:PuiChing_Macau/Collaborations">Team Macau</a></li>
 
−
                              <li><a href="https://2019.igem.org/Team:Tartu_TUIT/Collaboration">FAU-Tartz-TUIT</a></li>
 
−
                              <li><a href="(https://2019.igem.org/Team:FAU_Erlangen/Collaborations">Team
 
−
                                  FAU-Erlangen</a>
 
−
                              </li>
 
−
                              <li><a href="https://2019.igem.org/Team:Queens_Canada/Collaborations">Team
 
−
                                  Quens-Canada</a>
 
−
                              </li>
 
−
                            </ul>
 
−
                          </div>
 
−
                        </div>
 
−
                        <figure style="text-align: center; margin-left: 25px;">
 
−
                          <img src="https://static.igem.org/mediawiki/2019/f/f1/T--marburg--Doulix_webinar.jpeg"
 
−
                            alt="Bilder Webinar" style="max-height: 600px;">
 
−
                          <figcaption>
 
−
                            Figure 3: Participants watching our Golden Gate webinar all over the world.
 
−
                          </figcaption>
 
−
                        </figure>
 
−
                      </div>
 
−
                    </div>
 
−
                  </div>
 
−
                </div>
 
−
                <div class="wrap-collabsible" style="margin-bottom: 25px;">
 
−
                  <input id="collapsible2_2" class="toggle" type="checkbox">
 
−
                  <label for="collapsible2_2" class="lbl-toggle">
 
−
                    Slack Channel and gDrive - Global networking in the Golden Gate community
 
−
                    <hr style="width: unset;">
 
−
                  </label>
 
−
                  <div class="collapsible-content">
 
−
                    <div class="content-inner" style="text-align: left; text-align-last: left;">
 
−
                      <p style="margin-bottom: 25px;">
 
−
                        Although we were quite pleased with the result of our webinar, we thought about how to
 
−
                        further connect this Golden Gate community, so we created a Slack workspace for everyone
 
−
                        that is working with this method, enabling us all to exchange protocols and ideas or
 
−
                        troubleshoot issues that might arise. So far there are close to 50 members, but we hope
 
−
                        to find more enthusiasts willing to share their knowledge!<br>
 
−
                        All files used for the webinar are openly available. You can find our protocols,
 
−
                        handouts, slides as well as the files we used for the practical part in Geneious to
 
−
                        construct parts in-silico in our shared Google Drive. Through this we hope to make it
 
−
                        easier for other teams willing to start with Golden Gate cloning, as they can start
 
−
                        designing their first own in-silico parts with our guidance.
 
−
                      </p>
 
−
                      <p style="margin-bottom: 25px;">
 
−
                        The main documents of the Golgen Gate Webinar<br>
 
−
                        → Download: <a
 
−
                          href="https://static.igem.org/mediawiki/2019/0/0a/T--marburg--GG_webinar_slides.pdf">Golden
 
−
                          Gate webinar slide 2019</a><br>
 
−
                        → Download:<a
 
−
                          href="https://static.igem.org/mediawiki/2019/0/09/T--CMUQ--goldenGateWebinarHandout.pdf">Handout
 
−
                          Golden Gate webinar</a><br>
 
−
                        → Download:<a
 
−
                          href="https://static.igem.org/mediawiki/2019/c/c2/T--marburg--GG_protocoll_white.pdf">Golden
 
−
                          Gate protocoll</a>
 
−
                      </p>
 
−
                      <p>
 
−
                        To try out the described in-silico cloning you can download the needed sequences as
 
−
                        geneious file here.<br>
 
−
                        → Download:<a href="https://2019.igem.org/File:T--marburg--Sequences_Webinar.zip">Sequences
 
−
                          Webinar </a>
 
−
                      </p>
 
−
                    </div>
 
−
                  </div>
 
−
                </div>
 
−
                <div class="wrap-collabsible" style="margin-bottom: 25px;">
 
−
                  <input id="collapsible2_3" class="toggle" type="checkbox">
 
−
                  <label for="collapsible2_3" class="lbl-toggle">
 
−
                    Interlab - Accelerate Golden Gate Assembly
 
−
                    <hr style="width: unset;">
 
−
                  </label>
 
−
                  <div class="collapsible-content">
 
−
                    <div class="content-inner" style="text-align: left;">
 
−
                      <h2 class="subtitle">Interlab - Accelerate Golden Gate Assembly</h2>
 
−
                      <div style="display: flex; flex-direction: row;">
 
−
                        <p>
 
−
                          Interlab - Accelerate Golden Gate Assembly
 
−
                          As we are constantly working on improving the efficiency of Golden Gate. We clearly
 
−
                          saw the main bottleneck of the reaction was the long assembly time in the cycler.
 
−
                          In the webinar and in ourGolden Gate community weprovided protocols with three different
 
−
                          cycler settings for different applications.
 
−
                          Although these threeprotocols were tested before, they were never compared to each other in
 
−
                          their efficiency.<br>
 
−
                          We conducted our own Interlab study in collaboration with the iGEM Teams from
 
−
                          <a style="padding: 0;" href="https://2019.igem.org/Team:TUDelft"> TUDelft </a>,
 
−
                          <a style="padding: 0;" href="https://2019.igem.org/Team:Humboldt_Berlin"> HUmboldt Berlin </a>
 
−
                          and
 
−
                          <a style="padding: 0;" href="https://2019.igem.org/Team:TU_Kaiserslautern"> TU Kaiserslautern
 
−
                          </a>
 
−
                          In order to find the best protocol, we supplied the teams with specific Level 0
 
−
                          parts,that should be assembled to a Level 1 vector using the three different cycler
 
−
                          conditions. The provided parts areshown in the table
 
−
                        </p>
 
−
                        <div style="display: flex; flex-direction: column; margin-left: 25px;">
 
−
                          <figure style="float:left;">
 
−
                            <img src="https://static.igem.org/mediawiki/2019/7/79/T--marburg--interlab_button.svg"
 
−
                              alt="Interlab button" style="max-height: 200px;">
 
−
                          </figure>
 
−
                          <table style="min-width: 400px;">
 
−
                            <tbody>
 
−
                              <tr>
 
−
                                <td style="text-align: center">5' Connector </td>
 
−
                                <td style="text-align: center"> <a href="link 1"> BBa_K2560075 </a>/
 
−
                              </tr>
 
−
                              <tr>
 
−
                                <td style="text-align: center">Promoter </td>
 
−
                                <td style="text-align: center"> <a href="link 2"> J23119 BBa_K2560031 </a>
 
−
                              </tr>
 
−
                              <tr>
 
−
                                <td style="text-align: center">RBS </td>
 
−
                                <td style="text-align: center"> <a href="link 2"> B0034 BBa_K2560008 </a>
 
−
                              </tr>
 
−
                              <tr>
 
−
                                <td style="text-align: center">CDS </td>
 
−
                                <td style="text-align: center"> <a href="link 3"> sfGFP BBa_K2560042 </a>
 
−
                              </tr>
 
−
                              <tr>
 
−
                                <td style="text-align: center">Terminator </td>
 
−
                                <td style="text-align: center"> <a href="link 4"> B0015 BBa_K2560035 </a>
 
−
                              </tr>
 
−
                              <tr>
 
−
                                <td style="text-align: center">3' Connector </td>
 
−
                                <td style="text-align: center"> <a href="link 5"> BBa_K2560080 </a>
 
−
                              </tr>
 
−
                              <tr>
 
−
                                <td style="text-align: center">ORI: </td>
 
−
                                <td style="text-align: center"> <a href="link 6"> ColE1 BBa_K2560036 </a>
 
−
                              </tr>
 
−
                              <tr>
 
−
                                <td style="text-align: center">Antibiotic-Resistance-Cassette</td>
 
−
                                <td style="text-align: center"> <a href="link 7"> Kan-RFP BBa_K2560056 </a>
 
−
                              </tr>
 
−
                            </tbody>
 
−
                          </table>
 
−
                        </div>
 
−
                      </div>
 
−
                      <div style="display: flex; flex-direction: row;">
 
−
                        <figure style="text-align: center; margin-right: 25px">
 
−
                          <img src="https://static.igem.org/mediawiki/2019/9/91/T--marburg--interlab_package.jpeg"
 
−
                            alt="Interlab oackage">
 
−
                          <figcaption>
 
−
                            Fig 1: Interlab package.
 
−
                          </figcaption>
 
−
                        </figure>
 
−
                        <p>
 
−
                          The Golden Gate reactions should be pipetted together using the given parts and then
 
−
                          betransformed in E. coliand be plated with similar amounts on agar plates. A colony with a
 
−
                          correct assembled
 
−
                          vector would have afunctioning Transcription Unit which expresses GFP, which can be clearly
 
−
                          distinguished from RFP expressing colonies which only carries the antibiotic cassette and
 
−
                          false non fluorescent
 
−
                          colonies.<br>
 
−
                          The ratio of green to non-green colonies was used to evaluate which of the three protocols
 
−
                          yielded in the
 
−
                          highest transformation efficiency and the highest rate of positive clones.
 
−
                        </p>
 
−
                      </div>
 
−
                      <p>
 
−
                        The handout with the used protocols can be downloaded <a
 
−
                          href=https://static.igem.org/mediawiki/2019/a/a0/T--marburg--Interlab_protocoll.pdf>here </a>. </p>
 
−
                          <p>
 
−
                              <br>
 
−
                              <b>results</b>
 
−
                              <br>
 
−
                              First we wanted to know which protocol worked best for every team. For this we analysed the data of the correct assembled green colonies for every protocol. The data of the different teams is shown in table 1. The total colony count is hard to compare directly, cause the numbers differ very much between the teams. This could come through the different transformation efficiencies for example because of the variating competent cells the teams used.<br>
 
−
                              <br>
 
−
                             
 
−
 
−
                          </p>
 
−
                          </div> </div> </div> </section> </div> </div> </div>
 
−
                        <div class="sub" onclick="popup('rbn3')">
 
−
                          <div class="sub-header">
 
−
                            <h1>B E R L I N</h1>
 
−
                            <hr>
 
−
                          </div>
 
−
                          <div class="sub-content">
 
−
                            <img src="https://static.igem.org/mediawiki/2019/9/9b/T--marburg--berlin_logo_richtig.png">
 
−
                          </div>
 
−
                         
 
−
                    </div>
 
−
                    <div id="rbn3" class="popup">
 
−
                      <div class="popup-container">
 
−
                        <div class="popup-header">
 
−
                          <h1 class="title">Berlin Collaboration</h1>
 
−
                          <button type="button" onclick="hide('rbn3')">X</button>
 
−
                        </div>
 
−
                        <div class="popup-content" style="text-align: justify;">
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <p>
 
−
                              iGEM Berlin engineered the freshwater algae Chlamydomonas reinhardtii to degrade
 
−
                              Polyethylene
 
−
                              terephthalate
 
−
                              (PET). For this goal they also built a bioreactor for their chassis. We sent
 
−
                              them our UTEX
 
−
                              strain to test if
 
−
                              their photobioreactor is capable to cultivate other phototrophic organisms as
 
−
                              well. They gained
 
−
                              additional
 
−
                              experiences on growth conditions for phototrophic organisms and we could see our
 
−
                              strains grow in
 
−
                              abioreactor environment.<br>
 
−
                              Click here to learn more about there project <a
 
−
                                href="https://2019.igem.org/Team:Humboldt_Berlin">Chlamylicious</a>
 
−
                            </p>
 
−
                            <figure style="float:right; margin-left: 25px;">
 
−
                              <img src="https://static.igem.org/mediawiki/2019/9/9b/T--marburg--berlin_logo_richtig.png"
 
−
                                alt="Berlin logo">
 
−
                            </figure>
 
−
                          </div>
 
−
                        </div>
 
−
                      </div>
 
−
                    </div>
 
−
                    <div class="sub" onclick="popup('delft')">
 
−
                      <div class="sub-header">
 
−
                        <h1>D E L F T</h1>
 
−
                        <hr>
 
−
                      </div>
 
−
                      <div class="sub-content">
 
−
                        <img src="https://static.igem.org/mediawiki/2019/d/d4/T--marburg--DELF_logo_richtig.svg">
 
−
                      </div>
 
−
                    </div>
 
−
                    <div id="delft" class="popup">
 
−
                      <div class="popup-container">
 
−
                        <div class="popup-header">
 
−
                          <h1 class="title">Delft Collaboration</h1>
 
−
                          <button type="button" onclick="hide('delft')">X</button>
 
−
                        </div>
 
−
                        <div class="popup-content" style="text-align: justify;">
 
−
                          <div style="display: flex; flex-direction: row; margin-top: 25px;">
 
−
                            <figure style="margin-right: 25px;">
 
−
                              <img src="https://static.igem.org/mediawiki/2019/5/5e/T--marburg--Skype_DELF.jpeg" alt=" DELFT Bild"
 
−
                                style="max-width: 300px;">
 
−
                              <figcaption> Fig. 1: Skype talk with TU Delft</figcaption>
 
−
                            </figure>
 
−
                            <div style="display: flex; flex-direction: column;">
 
−
                              <div>
 
−
                                <h2 class="subtitle">Delft Collaborations</h2>
 
−
                                <p>
 
−
                                  iGEM Delft created an all species encompassing toolbox, based on orthogonal gene
 
−
                                  expression and
 
−
                                  host
 
−
                                  independent plasmid replication. We exchanged multiple times over Molecular
 
−
                                  Cloning and toolbox
 
−
                                  designs.
 
−
                                  Additionally, we sent them parts in the PhytoBricks standard.<br>
 
−
                                  Click here to learn more about there project <a href="https://2019.igem.org/Team:TUDelft">
 
−
                                    project SCI-PHI 29 </a>
 
−
                                </p>
 
−
                              </div>
 
−
                              <figure>
 
−
                                <img src="https://static.igem.org/mediawiki/2019/d/d4/T--marburg--DELF_logo_richtig.svg" alt="DELF logo"
 
−
                                  style="max-height: 200px; margin-top: 25px;">
 
−
                              </figure>
 
−
                            </div>
 
−
                          </div>
 
−
                        </div>
 
−
                      </div>
 
−
                    </div>
 
−
                    <div class="sub" onclick="popup('rbn4')">
 
−
                      <div class="sub-header">
 
−
                        <h1>
 
−
                          U C<span style="visibility: hidden;">A</span>D A V I S
 
−
                        </h1>
 
−
                        <hr>
 
−
                      </div>
 
−
                      <div class="sub-content" style="justify-content: center;">
 
−
                        <img style="max-height: 100%;" src="https://static.igem.org/mediawiki/2019/4/49/T--marburg--UCD_OT2.jpeg">
 
−
                      </div>
 
−
                    </div>
 
−
                    <div id="rbn4" class="popup">
 
−
                      <div class="popup-container">
 
−
                        <div class="popup-header">
 
−
                          <h1 class="title">UC Davis collaboration</h1>
 
−
                          <button type="button" onclick="hide('rbn4')">X</button>
 
−
                        </div>
 
−
                        <div class="popup-content">
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <figure Style="float:left; margin-right: 25px;">
 
−
                              <img src="https://static.igem.org/mediawiki/2019/4/49/T--marburg--UCD_OT2.jpeg" alt="UCD OT2">
 
−
                            </figure>
 
−
                            <div>
 
−
                              <p style="margin-bottom: 25px;">
 
−
                                UC Davis iGEM team is one of the winners of the Opentrons competition in 2019.
 
−
                                iGEM Marburg
 
−
                                won one OT-2
 
−
                                during last
 
−
                                year’s edition of the competition and got contacted for a collaboration by iGEM
 
−
                                UC Davis
 
−
                                along with other
 
−
                                winners from
 
−
                                2018 and 2019.
 
−
                                The goal for this automation InterLab collaboration was to collect data to
 
−
                                determine the
 
−
                                accuracy and
 
−
                                reproducibility of
 
−
                                the OT-2 compared to humans. To do this, UC Davis proposed to perform the
 
−
                                standard iGEM
 
−
                                protocol on serial
 
−
                                dilutions
 
−
                                with silica beads and fluorescein, each manually and with an OT-2. In total iGEM
 
−
                                Marburg did
 
−
                                four dilutions;
 
−
                                twice with
 
−
                                an OT-2 and twice manually. The data was then sent to UC Davis via the
 
−
                                spreadsheet designed
 
−
                                by them.
 
−
                                Finally, iGEM
 
−
                                Marburg aided by creating a Slack channel for all iGEM teams with an OT-2 to
 
−
                                facilitate
 
−
                                better communication
 
−
                                and
 
−
                                exchange in the future.
 
−
                              </p>
 
−
                              <p>
 
−
                                In total, seven teams participated and sent their data to UC Davis which are
 
−
                                shown in the
 
−
                                figure below.
 
−
                                According to UC
 
−
                                Davis, the fluorescence data was standardized using the iGEM MEFL conversion to
 
−
                                standardize
 
−
                                the plate reader
 
−
                                measurements. Subsequently, the data was fitted using a linear regression model,
 
−
                                whose
 
−
                                quality is quantified
 
−
                                using the
 
−
                                r2 metric (it ranges from 0 to 1 with higher value indicates a better fit).
 
−
                                <br>
 
−
                                For the fluorescence the r2 value from the OT2 data beats human r2 value with
 
−
                                0.94 and 0.90,
 
−
                                respectively.
 
−
                                Interestingly, for the silica beads, manual measurements yielded a more
 
−
                                satisfactory result
 
−
                                with r2 value of
 
−
                                0.90
 
−
                                compared to 0.82 from OT2 measurements. One possible explanation could be the
 
−
                                speed of the
 
−
                                OT-2: it took
 
−
                                twice as long
 
−
                                to complete this task with the OT-2 compared to a manual trial. This could lead
 
−
                                to the
 
−
                                sedimentation of the
 
−
                                silica beads
 
−
                                as the OT-2 went through their runs; therefore worsening its results. Also, as
 
−
                                shown in Fig.
 
−
                                2b, one team’s
 
−
                                result
 
−
                                deviates a lot from the rest of the teams, maybe if their result is taken from
 
−
                                the overall
 
−
                                evaluation a more
 
−
                                comparable
 
−
                                result between OT-2 and human can be observed.
 
−
                              </p>
 
−
                            </div>
 
−
                          </div>
 
−
                          <p style="margin-top: 25px;">
 
−
                            More information about this collaboration such as individual team data can be
 
−
                            found on UC
 
−
                            Davis wiki:
 
−
                            <a href="https://2019.igem.org/Team:UC_Davis/Collaborations"> Wiki UC Davis
 
−
                              Collaboration </a>
 
−
                          </p>
 
−
                          <figure style="text-align: center; margin-top: 25px;">
 
−
                            <img style="max-height: 300px"
 
−
                              src="https://static.igem.org/mediawiki/2019/c/cc/T--marburg--UC_Davis_Graphic_1.png"
 
−
                              alt=" UCD Graphic 1">
 
−
                            <img style="max-height: 300px"
 
−
                              src=" https://static.igem.org/mediawiki/2019/a/a7/T--marburg--UC_Davis_Graphic_2.png"
 
−
                              alt=" UC Davis Graphic 2">
 
−
                            <figcaption>
 
−
                              Figure 1: a)OT-2 vs manual fluorescein measurement experiment. OT-2
 
−
                              performed better
 
−
                              with r2 value of 0.94 compared to 0.90 of human. b)OT-2 vs manual silica
 
−
                              beads measurement experiment. Human performed better
 
−
                              with r2 value of 0.90 compared to 0.82 of OT-2.
 
−
                            </figcaption>
 
−
                          </figure>
 
−
                        </div>
 
−
                      </div>
 
−
                    </div>
 
−
                    <div class="sub" onclick="popup('rbn5')">
 
−
                      <div class="sub-header">
 
−
                        <h1>
 
−
                          U F L O R I D A
 
−
                        </h1>
 
−
                        <hr>
 
−
                      </div>
 
−
                      <div class="sub-content">
 
−
                        <img src="https://static.igem.org/mediawiki/2019/f/f7/T--Florida--iGEMWeb1.png">
 
−
                      </div>
 
−
                    </div>
 
−
                    <div id="rbn5" class="popup">
 
−
                      <div class="popup-container">
 
−
                        <div class="popup-header">
 
−
                          <h1 class="title">UFlorida</h1>
 
−
                          <button type="button" onclick="hide('rbn5')">X</button>
 
−
                        </div>
 
−
                        <div class="popup-content" style="text-align: justify">
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <figure style="float:left; margin-right: 25px;">
 
−
                              <img src="https://static.igem.org/mediawiki/2019/f/f7/T--Florida--iGEMWeb1.png" alt="Florida">
 
−
                            </figure>
 
−
                            <p>
 
−
                              This semester we had the pleasure of mentoring UFlorida for their Wiki
 
−
                              design. Our member, Rene Inckemann, aided UFlorida in their quest to create
 
−
                              a visually accessible Wiki, similar to what we did this past iGEM season.
 
−
                              We helped shape UFlorida’s website into an interactive and appealing
 
−
                              landscape, while maintaining the integrity of their goal and project. We
 
−
                              advised them on their font choices- specifically their use of bold and
 
−
                              italicized words-, along with answering questions about our work with the
 
−
                              visually disabled community and utilizing our “Accessible Wiki Guide” from
 
−
                              the previous year.
 
−
                              <br>
 
−
                              Besides answering questions specifically about their project, we also
 
−
                              offered valuable information about when to start working on the Wiki and
 
−
                              questions about coding. Working with a young team like UFlorida offered us
 
−
                              a great mentorship opportunity.
 
−
                            </p>
 
−
                          </div>
 
−
                          <figure style="align-content:center; margin-top: 25px;">
 
−
                            <img src="https://static.igem.org/mediawiki/2019/2/29/T--marburg--UFlorida_Rene.jpeg"
 
−
                              alt="Florida">
 
−
                            <figcaption>
 
−
                              Fig. 1: Skypecall with UFlorida
 
−
                            </figcaption>
 
−
                          </figure>
 
−
                        </div>
 
−
                      </div>
 
−
                    </div>
 
−
                    <div class="sub" onclick="popup('rbn6')">
 
−
                      <div class="sub-header">
 
−
                        <h1>I N T E R A C T I O N S</h1>
 
−
                        <h1>W I T H</h1>
 
−
                        <h1>O T H E R<span style="visibility: hidden;">A</span>T E A M S</h1>
 
−
                        <hr>
 
−
                      </div>
 
−
                      <div class="sub-content">
 
−
                        <img src="https://static.igem.org/mediawiki/2019/f/fe/T--marburg--Postcard.png">
 
−
                      </div>
 
−
                    </div>
 
−
                    <div id="rbn6" class="popup">
 
−
                      <div class="popup-container">
 
−
                        <div class="popup-header">
 
−
                          <h1 class="title">Interactions with other teams</h1>
 
−
                          <button type="button" onclick="hide('rbn6')">X</button>
 
−
                        </div>
 
−
                        <div class="popup-content" style="text-align: justify;">
 
−
                          <h2 class="subtitle">Mike the microbe</h2>
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <figure Style="text-align: center; margin-right: 25px;">
 
−
                              <img src="https://static.igem.org/mediawiki/2019/b/b0/T--marburg--Mike_microbe.png"
 
−
                                alt=" Mike the Mircrobe">
 
−
                              <figcaption>
 
−
                                Figure 1: Mike the mircrobe in our lab.
 
−
                              </figcaption>
 
−
                            </figure>
 
−
                            <p>
 
−
                              We participated in the “Mike the microbe” collaboration of the iGEM Team US AFRL
 
−
                              CarrollHS.
 
−
                              They
 
−
                              sent us
 
−
                              their virtual mascots Mike the Microbe and Chia the Chitinase so we could take pictures
 
−
                              with
 
−
                              them to
 
−
                              connect
 
−
                              all iGEM teams around the world via social media. It brought us great joy to join to this
 
−
                              collaboration and
 
−
                              let the two little buddies visit our daily life in our incubators for cyanobacteria.
 
−
                            </p>
 
−
                          </div>
 
−
                          <h2 class="subtitle" style="margin-top: 25px;">Postcards Düsseldorf</h2>
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <p>
 
−
                              iGEM Düsseldorf called for their traditional collaboration project, a long year postcard
 
−
                              campaign with the
 
−
                              purpose to educate and to show the public about the different topics and projects in
 
−
                              Synthetic
 
−
                              Biology and
 
−
                              especially regarding all the iGEM projects. With interesting designs and a short project
 
−
                              abstract iGEM teams
 
−
                              could spread their ideas using the postcards to share their thinking to each other and the
 
−
                              society. Our
 
−
                              title and postcard design was about our photosynthetic project „Syntex“. You can see
 
−
                              Synechococcus elongatus
 
−
                              UTEX 2973 direly needing a refreshing drink, a cute little draft from our team member
 
−
                              Jonas.
 
−
                            </p>
 
−
                            <figure style="text-align:center; margin-left: 25px;">
 
−
                              <img src="https://static.igem.org/mediawiki/2019/f/fe/T--marburg--Postcard.png"
 
−
                                alt="Postcard challenge">
 
−
                              <figcaption>
 
−
                                Figure 2: Our Postcard for the Postcard collaboration from Düsseldorf.
 
−
                              </figcaption>
 
−
                            </figure>
 
−
                          </div>
 
−
                          <h2 class="subtitle" style="margin-top: 25px;">iGEMxSDGs challenge</h2>
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <figure style="text-align:center; margin-right: 25px;">
 
−
                              <img src="https://static.igem.org/mediawiki/2019/1/18/T--marburg--SDG_chalenge.png"
 
−
                                alt="SGD Challenge">
 
−
                              <figcaption>
 
−
                                Figure 3: SDG challenge.
 
−
                              </figcaption>
 
−
                            </figure>
 
−
                            <p>
 
−
                              IGEM Costa Rica, Tuebingen, and TAS Taipei created an iGEMxSDGs challenge as
 
−
                              collaboration.
 
−
                              They
 
−
                              asked us to
 
−
                              choose four of the Sustainable Development Goals (SDGs) proposed by the United Nations
 
−
                              (UN).
 
−
                              We
 
−
                              joined this
 
−
                              collaboration challenge and spread it on our social media account. We decided on the
 
−
                              following
 
−
                              goals:
 
−
                            </p>
 
−
                          </div>
 
−
                          <h2 class="subtitle" style="margin-top: 25px;">Who wore it best? #labcoatchallenge</h2>
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <p>
 
−
                              The Stony Brook iGEM Team challenged us to design an outfit for a runway from a standard
 
−
                              lab
 
−
                              coat.
 
−
                              The
 
−
                              outfit had to be created by tying, not using duct tape, cut or sew, etc. After designing
 
−
                              our
 
−
                              outfits
 
−
                              four of
 
−
                              our members presented their style on our ‘runway’ while paparazzi took their photos. As an
 
−
                              outfit we
 
−
                              chose a
 
−
                              dress highlighting the neckline, a shoulder free dress, a knotted dress and a skirt to
 
−
                              bring
 
−
                              glamour
 
−
                              to the
 
−
                              daily routine in the labs. Especially our guys were rocking their outfits as you can see
 
−
                              here
 
−
                            </p>
 
−
                            <figure style="text-align:center; margin-left: 25px;">
 
−
                              <img src="https://static.igem.org/mediawiki/2019/7/74/T--marburg--Labcoat_Burgi.jpeg"
 
−
                                alt="labcoat">
 
−
                              <figcaption>
 
−
                                Figure 4: Our model Burgis for the labcoatchallenge.
 
−
                              </figcaption>
 
−
                            </figure>
 
−
                          </div>
 
−
                        </div>
 
−
                      </div>
 
−
                    </div>
 
−
                    <div class="sub" onclick="popup('rbn7')">
 
−
                      <div class="sub-header">
 
−
                        <h1>M E E T U P S</h1>
 
−
                        <hr>
 
−
                      </div>
 
−
                      <div class="sub-content">
 
−
                        <img src="https://static.igem.org/mediawiki/2019/9/90/T--Marburg--meetups.jpg">
 
−
                      </div>
 
−
                    </div>
 
−
                    <div id="rbn7" class="popup">
 
−
                      <div class="popup-container">
 
−
                        <div class="popup-header">
 
−
                          <h1 class="title">Meet ups</h1>
 
−
                          <button type="button" onclick="hide('rbn7')">X</button>
 
−
                        </div>
 
−
                        <div class="popup-content" style="text-align: justify;">
 
−
                          <h2 class="subtitle">iGEM Spring Festival in Bonn</h2>
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <p>
 
−
                              From 3rd – 5th Mai we met our iGEM colleagues for the Spring Festival in Bonn. Professors
 
−
                              of
 
−
                              the
 
−
                              university of Bonn presented their research fields which gained huge interest. We heard
 
−
                              lectures
 
−
                              of immunology and neurosciences, as well as reviving mammoths. During the lectures,
 
−
                              specific
 
−
                              topics were deepened in small seminar groups, such as the ethical tenability of CRISPR
 
−
                              applied
 
−
                              at humans and other animals. At the poster sessions we got the first time in contact with
 
−
                              the
 
−
                              other teams’ projects. Afterwards we had the opportunity to vote for the best project,
 
−
                              which
 
−
                              iGEM Düsseldorf won. Besides that, the meetup was filled with a lot of awarding
 
−
                              categories.
 
−
                              We
 
−
                              competed in the categories for “Best Video”, “Best Poster”, “Best Project” and a single
 
−
                              person
 
−
                              had the chance to win the award for “Best Meme”. The iGEM Team Bonn offered spontaneously
 
−
                              a
 
−
                              little football table tournament, where two of our team members won. In the evening we
 
−
                              learnt a
 
−
                              lot about Bonn as a city from a guide living there for ages. Afterwards, the iGEM Teams
 
−
                              met
 
−
                              in
 
−
                              several groups in restaurants and bars. We merged with iGEM Kaiserslautern and exchanged
 
−
                              our
 
−
                              projects and ideas for the rest of the evening.<br>
 
−
                              On the second day, the iGEM Team Bonn offered a competition out of three rounds, where we
 
−
                              competed against other teams. We were asked iGEM specific questions, had to solve riddles
 
−
                              for an
 
−
                              escape room and finished a lab challenge afterwards. Our team could compete until the end
 
−
                              and
 
−
                              won the prize! Thanks again to Doulix for this awesome prize!<br>
 
−
                              We particularly thank the iGEM Team Bonn for the huge effort and the great meetup!
 
−
                            </p>
 
−
                            <figure style="text-align:center; margin-left: 25px">
 
−
                              <img style="min-width: 300px;"
 
−
                                src="https://static.igem.org/mediawiki/2019/a/a9/T--marburg--meet_u_bonn.png"
 
−
                                alt="Erlangen meetup">
 
−
                              <figcaption>
 
−
                                Fig. 1: Two representatives of Doulix celebrating with us.
 
−
                              </figcaption>
 
−
                            </figure>
 
−
                          </div>
 
−
                          <h2 class="subtitle" style="margin-top: 25px;">Erlangen collaboration meetup</h2>
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <figure style="text-align:center; margin-right: 25px">
 
−
                              <img style="min-width: 300px;"
 
−
                                src="https://static.igem.org/mediawiki/2019/0/01/T--marburg--meet_up_erlangen.jpeg"
 
−
                                alt="Erlangen meetup">
 
−
                              <figcaption>
 
−
                                Fig. 2: Group photo from the Erlangen mett up (Michael Burgis from Team Marburg in the
 
−
                                left
 
−
                                side).
 
−
                              </figcaption>
 
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                            </figure>
 
−
                            <p>
 
−
                              On the 17th – 19th of May we attended the collaboration meetup hosted by the iGEM Team
 
−
                              Erlangen
 
−
                              2019. There, we met the iGEM teams of Straßbourg, Tübingen, Vienna and of course Erlangen.
 
−
                              Apart
 
−
                              from listening to the interesting talks from the various professors there, we made a lot
 
−
                              of
 
−
                              new
 
−
                              friends and took the chance to talk about possible collaborations with the other teams and
 
−
                              also
 
−
                              promote our collaborations, such as the colony picking project and the Golden Gate webinar
 
−
                              (evtl. verlinken). We would like to thank iGEM Team Erlangen very much for organizing this
 
−
                              wonderful meetup and are happy to see them soon in Boston.
 
−
                            </p>
 
−
                          </div>
 
−
                          <h2 class="subtitle" style="margin-top: 25px;">Düsseldorf meetup</h2>
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <p>
 
−
                              From 5th – 7th July we went to the HHU for the German meetup. The calendar offered lots
 
−
                              of
 
−
                              lectures with interesting topics such as the Excellent Cluster of Prof. Andreas Weber
 
−
                              “CEPLAS”
 
−
                              but also non-scientific aspects like how to do presentations. We tried to visit all of
 
−
                              them
 
−
                              while presenting our poster in the hall. There we got in touch with other iGEM Teams also
 
−
                              learnt
 
−
                              about their projects. We promoted our Webinar project and laid the foundation stone for
 
−
                              future
 
−
                              collaborations. On the second day, iGEM Düsseldorf organized a panel discussion about the
 
−
                              issues
 
−
                              concerning green genetic engineering. Thereby, we got great input on how to structure our
 
−
                              panel
 
−
                              discussion and which subjects should be mentioned. Beside the regular program organized by
 
−
                              iGEM
 
−
                              Düsseldorf we used a lot of chances to get in contact with other teams. After the
 
−
                              presentations
 
−
                              a couple of tourist guides showed us the beautiful old town of Düsseldorf and explained
 
−
                              some
 
−
                              interesting historical facts. Afterwards we had again the possibility to meet with other
 
−
                              iGEMers
 
−
                              in a pub and talk about the projects, especially to find weaknesses or opportunities for
 
−
                              collaborations. This really went successfully due to the time we spend with iGEM Team
 
−
                              Aachen
 
−
                              and
 
−
                              Kaiserslautern. The iGEM Team Düsseldorf also arranged a game contest between the teams,
 
−
                              where
 
−
                              we lost in the final to our good friends from Potsdam. Congrats! <br>
 
−
                              Thanks again to iGEM Düsseldorf for the huge effort and the great meetup!
 
−
                            </p>
 
−
                            <figure style="text-align:center; margin-left: 25px;">
 
−
                              <img style="min-width: 200px;"
 
−
                                src="https://static.igem.org/mediawiki/2019/e/e4/T--marburg--meet_up_Duesseldorf.jpeg"
 
−
                                alt="Du meetup">
 
−
                              <figcaption>
 
−
                                Fig. 3: Team photo from the Düsseldorf meetup.
 
−
                              </figcaption>
 
−
                            </figure>
 
−
                          </div>
 
−
                          <h2 class="subtitle" style="margin-top: 25px;">Vilnius, Lithuania – Biohackathon Lab App 2019
 
−
                          </h2>
 
−
                          <div style="display: flex; flex-direction: row;">
 
−
                            <!--<figure style="text-align:center; margin-right: 25px">
 
−
                  <img src="Pic" alt="Vilnius hacker">
 
−
                  <figcaption>
 
−
                    Fig. 4: Meet up Vilnius, Lithuania.
 
−
                  </figcaption>
 
−
                </figure>-->
 
−
                            <p>
 
−
                              In August 2019 our Team participated in the Biohackathon Lab App, organized by the
 
−
                              Vilnius-Lithuania Team 2019. Together with the iGEM Teams Stockholm and UCopenhagen, as
 
−
                              well
 
−
                              as
 
−
                              several non-iGEM Teams we sat together for a weekend packed with problems, ideas and neat
 
−
                              solutions.
 
−
                              With the task of creating an application that tackles a self-found problem, we sat
 
−
                              together
 
−
                              and
 
−
                              brainstormed about what could be done.
 
−
                              <br>
 
−
                              During this time we were starting to work more with the Opentrons OT-2 and soon realized
 
−
                              that
 
−
                              some
 
−
                              of our biologists had trouble integrating protocols in the robot themselves, as this is
 
−
                              primarily
 
−
                              done by writing the protocol in Python and pushing it directly to the robot – no
 
−
                              convenient
 
−
                              application to change parameters, such as the amount of reactions,was included in the
 
−
                              protocols.
 
−
                              In our heads the idea to have a user-friendly GUI grew more and more and soon we found
 
−
                              ourselves
 
−
                              standing in the Vilnius University Life Sciences Center.
 
−
                              <br>“We” were two biologists and one mathematician, so we talked to some of the other
 
−
                              participants
 
−
                              that did not have a team yet and found the perfect fit to complement our small team: Nour
 
−
                              Alsamsam
 
−
                              was happy to join us for this weekend and provided us with his coding skills – huge thank
 
−
                              you
 
−
                              to
 
−
                              you, Nour!
 
−
                              Together we tried our best to come up with a suitable app and presented it in the end,
 
−
                              having
 
−
                              more
 
−
                              ideas in our heads than we could implement in such a short time – it was clear for us,
 
−
                              that we
 
−
                              would continue with this project later on!
 
−
                              <br>In conclusion, the Biohackathon was an awesome and productive meeting of the iGEM
 
−
                              Teams
 
−
                              Stockholm, Copenhagen, Vilnius and Marburg. We talked about our projects, offered each
 
−
                              other
 
−
                              feedback on current issues in the lab and shared many ideas.
 
−
                              We are very grateful that we got the opportunity to meet so many experts from different
 
−
                              fields,
 
−
                              which really helped getting a fresh perspective on our project and are now looking forward
 
−
                              to
 
−
                              meet
 
−
                              the other teams again at the Giant Jamboree!
 
−
                            </p>
 
−
                          </div>
 
−
                        </div>
 
−
                      </div>
 
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                    </div>
 
−
                  </div>
 
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              </section>
 
 
             </div>
 
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Revision as of 01:35, 22 October 2019

I M P R O V E

Every Synbio Experiment is more or less based on the same principle: You change a system in some way and you look at the outcome. This readout is one of the most important things in all natural science, a wrong readout can easily flaw your whole experiment or can lead to serious misconclusion.
The most common way to measure localisation, interaction or even the intensity of genetic elements is via Fluorescence as readout.
Fluorescence Proteins (FP), started with the green fluorescent protein, are based on the ability of a chromophore to absorb photons of specific wavelength and emit this photon at another. Even on the iGEM registry, the characterization via FPs is the suggested way to characterise a part.
This Method is prone to Background noise, depends on the folding of the Protein at the specific cell conditions and furthermore the chromophore can even bleach after to much exposure, so the drawbacks are obvious.

Bioluminescence could make the desired difference, but the original Luciferase Assays either consistent of an whole Operon systems, or put an unnecessary high metabolic burden through ATP dependency and/or trough its relatively large size (Firefly-Luciferase 61,5 kDa). Together with the low quantity, which can be several orders of magnitude lower than a fluorescence based system, the common breakthrough of Lumincese in Synthetic biology is still missing.
Newly developed small ATP independent Lucferase Proteins, are interesting candidates to bypass these Problems. Nanoluc, with its 19 kDa and up to 150 fold increase in brightness compared to the Firefly-Luciferase is handled as an suitable alternative. This Protein use the patented Substrate Furimazine, and emits Photons at 460 nm. Naoluc has been successfully implemented in Promoter testing and as an alternative in Interaction messurement via Bilumiecnce Resonace energy transfer, but sadly only few team ever used this system.
One scratch on the surface of Nanoluc is for sure the restriction of the wavelength. While for Measurements in many organisms and Tissues, this looming Problem did not occur, it's becoming obvious, when looking into phototrophic Organisms and deep-tissue mammalian cells. As the keen reader might guess, cells absorb Light of the wavelength under 600 nm to a great extent and even more if they have a photosystem. Chlorophyll a have one their two peaks at 440 nm [fig.1]. If one would compare that with the nanoluc spectra, a devastating conclusion could be made: The Photosystem will absorb photons from the Signal, leading to weaker peaks, and maybe more grave/frightening/alarming, a dependency of Signal on the chlorophyll content. Althroug localisation experiments should´t be affected that much, Measurement and characterisation, the foundation of which synthetic Biology is build on, could be shaken. Driven by this problem, we dig ourselves in literature and found a solution: A mutated Version of nanoLuc, so called teLuc
BBa_BBa K3228042 BBa K3228042
which has a severe red shifted pattern with a peak at 502 nm (Figure 2). What is even more serviere is the astonishing brightness, wich even surpass nanoluc by several folds (5,7) in vitro. In vivo this effect is even more dramatic, through its ability to bypass the absorption of Light. We expect this ability of teLuc to surpass the limits of Luminescence in plants to an amazing extent, and allow the plant synthetic biology community to accelerate their research.

Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra
Fig.2 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra

Lvl1 ori
Fig.1 - Lvl1 ori

Introduction of exogenous DNA can be done in multiple ways and propagated in a strain if it is integrated in the chromosome or stably expressed on a self-replicating plasmid.
For rapid prototyping in cyanobacteria self-replicating plasmids are of higher interest than genome-integrations, as the latter can be quite time-consuming in cyanobacterial strains with multiple genome copies (Griese et al., 2011). Furthermore, genes introduced in self-replicating vectors have been shown to have higher gene-expression levels than those integrated in the genome, as copy numbers are typically higher (Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2016) – a desirable trait, not just for rapid prototyping in research applications, but also for biotechnological production of valuable compounds.
With our shuttle-vectors encompass a cyanobacterial origin of replication (ori) from Synechococcus elongatus PCC7942 as well as an E.coli ori, which is perfect for fast cloning processes, as these vectors can be easily recovered from the cyanobacteria and reintroduced in an E.coli strain.


Currently existing shuttle vectors for cyanobacteria are still based on standard systems working with multiple cloning sites (MCS) for expression of homologous genes (Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2016). A huge downside is that these vectors include either an MCS (e.g. pAM5188) or a fluorescence reporter (e.g. pAM4787), which is unpractical for easy selection of recombinant clones. Additionally, an MCS comes with possible sequence constraints due to restriction sites leaving unwanted base pairs in your constructs.
Facilitating and standardizing the process of engineering biological systems is one of the fundamental goals of synthetic biology (Shetty Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2008), so the construction of a shuttle-vector based on a modular cloning method significantly improves the genetic toolbox we created for genetic engineering and synthetic biology approaches in S.elongatus and other cyanobacteria.


The commonly used S.elongatus strain PCC7942 carries two endogenous plasmids, the 46,4kb pANL (Chen et al., 2008) which is essential and the 7,8kb pANS (Van der Plas et al., 1992) which is not essential for the strain and can easily be cured.
This small plasmid has already been used for construction of shuttle vectors (Kuhlemeier Titel anhand dieser DOI in Citavi-Projekt übernehmen & van Arkel, 1987 ; Golden & Sherman, 1983 ; Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2016).
We followed this lead to create the best shuttle-vector available for cyanobacteria by encompassing the minimal replication region of pANS and the ColE1 origin of replication into our vectors, allowing for stable self-replication with high copy numbers in cyanobacteria (Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2016) and E.coli (Gerhart Titel anhand dieser DOI in Citavi-Projekt übernehmen et al.,2002). This addition to the genetic toolbox proves invaluable, as it can be easily recovered from the cyanobacterial strain and reintroduced in E.coli for fast GoldenGate-based cloning processes.


In order to supply the community with an easy selection system, we equipped our shuttle vector with a fluorescent reporter that is cut out when introducing new genetic parts:
A mRFP (red fluorescent protein) cassette is flanked by our standardized TypeIIS restriction enzyme recognition sequences (BsmBI or BsaI depending on what level you want to clone in). In a standard Golden Gate reaction this cassette will drop out and leave space for the parts that should be introduced, allowing for easy selection on plate after successful cloning – red colonies are wrong, still harboring the mRFP cassette and white colonies (if no other fluorescence is introduced) are correct, as the mRFP was switched with the parts of interest.

This crucial part comes in two variations - one for cloning Lvl1 and one for Lvl2 constructs -, giving the Golden Gate community everything they need for successful and reliable creation of self-replicating vectors in cyanobacteria.