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| − | {{Marburg}}
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| − | <div>
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| − | <div class="box-dark">
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| − | <h1 class="heading">
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| − | L A B A U T O M A T I O N
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| − | </h1>
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| − | <hr class="line">
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| − | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg"
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| − | class="logo"
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| − | alt="Syntex Logo">
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| − | </div>
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| − | <div style="margin-top: 10vh;">
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| − | <section class="section">
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| − | <h1 class="title">Amplifying new standards in measurement</h1>
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| − | <p style="text-align: justify;">
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| − | This year’s iGEM Team worked extensively on automating a plasmid purification on Opentrons’ OT-2. Plasmid
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| − | purification is an indispensable part of completing the cloning workflow in the OT-2.<br>
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| − | <br>
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| − | </p>
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| − | <figure style="float: right; margin-left: 25px;">
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| − | <img style="height: 400px; width: 600px"
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| − | src="https://static.igem.org/mediawiki/2019/6/60/T--Marburg--SyntexConnections.png"
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| − | alt="Connections between Opentrons, Promega and QInstruments">
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| − | <figcaption style="max-width: 600px">
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| − | Fig.1 - iGEM team Marburg 2019 is establishing connections between Opentrons, Promega and QInstruments.
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| − | </figcaption>
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| − | </figure>
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| − | <div><p>
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| − | Since the time of an iGEM project is limited to only one year, consequently only a limited amount of work can be
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| − | done in that time, which is even reduced by failing experiments and making mistakes in the lab. To overcome this
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| − | problem and increase the reproducibility and simultaneously raise the amount of experiments in the lab, we
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| − | automated plasmid purification on the OT-2. Using this protocol and making it open-source <b>(GitHub Link?)</b>,
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| − | we
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| − | achieved to parallelize work in the lab or make more time for public engagement, human practice, IHP or
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| − | everything else not directly lab-related, benefiting the whole iGEM community. This benefits will also be
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| − | translated beyond iGEM community such as in the amateur biohackers, enthusiasts, and students community and even
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| − | to research groups doing cutting-edge research.<br>
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| − | This idea started when we found out that there is also a great need in the industry for an automated cloning
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| − | workflow. Promega provided us with great advice <b>(Link to IHP)</b> and sponsored the Wizard® MagneSil® Plasmid
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| − | Purification System, QInstruments sponsored the BioShake D30-T elm and Opentrons sponsored their Magnetic
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| − | Module. Through our work aligned with the philosophy of iGEM for nurturing collaborations, we enabled
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| − | connections between these companies to achieve the true potential of their products. This kind of bridge would
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| − | not have been possible otherwise.<br>
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| − | <br>
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| − | Nevertheless, a massive amount of barriers had to be broken down. The shaker was a bit bigger than the space
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| − | normally occupied by modules in the OT-2 and needed stabilizing support, so it was obvious to design a
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| − | custom-made shaker adapter and print it with our own in-house 3D printer, which would keep the costs for the
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| − | automation of this workflow extremely low. Moreover, the 3D design will be publicly available in our GitHub
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| − | repository (LINK), which will make our solution accessible to everyone with access to a 3D printer.<br>
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| − | <br>
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| − | Additionally, we stumbled across serious problems with the calibration of our OT-2 and accessing the shaker with
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| − | the pipette. The BioShake D30-T elm is currently not a usual labware defined by Opentrons’, so we had to be
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| − | creative and come up with our own labware definition. Opentron is recently rolling out a major update from their
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| − | OT-2 3.9 to 4.0 firmware that includes a lot of paradigm changes, making it impossible for us to define it as a
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| − | decent custom labware. That is why we came up with the idea to use Opentrons’ internal coordinate system and
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| − | defining the required 96 Deep Well Plate on the shaker as coordinates. This facilitated accessing the shaker
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| − | with the pipette, being as precise as Opentrons’ own labware definitions, but a whole series of problems
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| − | followed, as we tried to use Opentrons’ pipette functions to transfer the chemicals. We managed these problems
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| − | as well, by defining our own Python functions, telling the pipette how to transfer liquids from and to the
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| − | defined shaker. In the end when running the script, one would not be able to tell the difference between the
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| − | labware and functions defined by us from the ones defined by Opentrons’.<br>
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| − | <br>
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| − | </p>
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| − | <figure align=center>
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| − | <img style="height: 500px; width: 300px"
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| − | src="https://static.igem.org/mediawiki/2019/b/bb/T--Marburg--opentrons_magnetic_module.JPG"
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| − | alt="OT-2 left">
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| − | <img style="height: 500px; width: 300px"
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| − | src="https://static.igem.org/mediawiki/2019/3/30/T--Marburg--opentrons_shaker.JPG" alt="OT-2 right">
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| − | <figcaption style="max-width: 1400px">
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| − | Fig.2 - Single-Channel pipette, magnetic module and shaker in action while performing the plasmid
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| − | purification.
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| − | </figcaption>
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| − | </figure>
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| − | <br>
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| − | <p>
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| − | Putting the pieces together, we were able to translate the manual plasmid purification protocol provided by Nans
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| − | Bodet into an Opentrons protocol, being the very first of its kind. We pioneered a workflow for up to six
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| − | samples with the p300 Single-Channel Electronic Pipette and a scaled-up version for up to 48 samples with the
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| − | p300 8-Channel Electronic Pipette without having to intervene even once. This scalability provides important
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| − | flexibility for various kinds of experiments.<br>
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| − | <br>
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| − | In our process of developing and running the protocol we determined some problems on increasing the yield of our
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| − | plasmids. There was a large number of parameters that could be varied, changing the final concentration of the
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| − | plasmids. For example, we realized that the duration of lysis is paramount for the yield and success of plasmid
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| − | purification. Over-lysis will lead to a decrease in plasmid yield, whereas under-lysis will induce clumping of
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| − | magnetic beads; thus failing the experiment. After a whole heap of plasmid purifications we managed to identify
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| − | the most relevant parameters and improve the protocol in the best way possible.<br>
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| − | <br>
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| − | </p>
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| − | <figure align=center>
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| − | <img style="height: 700px; width: 600px"
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| − | src="https://static.igem.org/mediawiki/2019/e/ea/T--Marburg--SingleChannelSetup.png" alt="OT-Layout left">
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| − | <img style="height: 700px; width: 600px"
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| − | src="https://static.igem.org/mediawiki/2019/d/df/T--Marburg--8channelSetup.png" alt="OT-Layout right">
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| − | <figcaption style="max-width: 1400px">
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| − | Fig.3 - Final setup for the automated plasmid purification workflows in the OT-2. The left picture shows the
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| − | setup for the single channel workflow, the right picture for the 8-channel workflow.
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <video src="https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--PlasmidPurificationMarburg.mp4" controls
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| − | poster="vorschaubild.jpg"></video>
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| − |
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| − | <br>
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| − | </p>
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| − | </section>
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| − | <section class="section">
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| − | <article>
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| − | <h1 class="title"></h1>
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| − | <p style="text-align: justify; margin-bottom: 1em;">
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| − |
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| − | </p></div>
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| − | </article>
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| − | </section>
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| − | <hr>
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| − | </div>
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| − | </div>
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| − | </body>
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| − | </html>
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