Difference between revisions of "Team:Marburg/test joana"

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         M I N I P R E P
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         P A R T S O V E R V I E W
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        <h1 class="title">A.P.P Automated Purfication Protocolment</h1>
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                    <h1 class="title">Parts Overview</h1>
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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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                     With our additional created parts, we expanded the current Marburg Collection.
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                     src="https://static.igem.org/mediawiki/2019/6/60/T--Marburg--SyntexConnections.png"
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                     <br>
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                    alt="Connections between Opentrons, Promega and QInstruments">
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                    <br>
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                 </p>
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                     Fig.1 - iGEM team Marburg 2019 is establishing connections between Opentrons, Promega and QInstruments.
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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 <b><a
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                     href="https://github.com/igemsoftware2019/iGemMarburg2019">open-source</a></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>
 
                 <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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                        <article class="media">
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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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                            <div class="media-content">
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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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                                    <h2>Best Basic Part</h2>
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                            <h1>Best Basic Part</h1>
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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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                        <article class="media">
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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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                                    <h2>Best Composite Part</h2>
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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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                                    <p>
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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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                                        1-2 sentence abstract
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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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                            <h1>Best Composite Part</h1>
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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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                                    <h2>Marburg Collection 2.0</h2>
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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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                                        1-2 sentence abstract
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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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                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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                            <h1>Marburg Collection 2.0</h1>
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                flexibility for various kinds of experiments.<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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                            <p>
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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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                                Parts of the Marburg Collection 2.0
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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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                                <br><br>
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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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                                Download the "Marburg Collection 2.0" (als downloadlinkg und symbol zum download dazu)
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                the most relevant parameters and improve the protocol in the best way possible.<br>
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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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                <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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            <video src="https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--PlasmidPurificationMarburg.mp4" controls
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Revision as of 22:57, 21 October 2019

P A R T S O V E R V I E W

Parts Overview



With our additional created parts, we expanded the current Marburg Collection.


Best Basic Part

1-2 sentence abstract


Best Composite Part

1-2 sentence abstract


Marburg Collection 2.0

1-2 sentence abstract