Team:Marburg/Miniprep
M I N I P R E P
A.P.P. Automated Purfication Protocol
This year’s Marburg team worked extensively on automating a plasmid purification on Opentrons’ OT-2. Plasmid
purification is an indispensable part of completing the cloning workflow in the OT-2.
Since the time of an iGEM project is limited to only one year, consequently only a limited amount of work can be
done in that time, which is even reduced by failing experiments and making mistakes in the lab. To overcome this
problem and increase the reproducibility and simultaneously raise the amount of experiments in the lab, we
automated plasmid purification on the OT-2. Using this protocol and making it open-source,
we
achieved to parallelize work in the lab or make more time for public engagement, human practice, IHP or
everything else not directly lab-related, benefiting the whole iGEM community. This benefits will also be
translated beyond iGEM community such as in the amateur biohackers, enthusiasts, and students community and even
to research groups doing cutting-edge research.
This idea started when we found out that there is also a great need in the industry for an automated cloning
workflow. Promega provided us with great advice and sponsored the Wizard® MagneSil® Plasmid
Purification System, QInstruments sponsored the BioShake D30-T elm and Opentrons sponsored their Magnetic
Module. Through our work aligned with the philosophy of iGEM for nurturing collaborations, we enabled
connections between these companies to achieve the true potential of their products. This kind of bridge would
not have been possible otherwise.
Nevertheless, there were many challenges. The shaker was a bit bigger than the space
normally occupied by modules in the OT-2 and needed stabilizing support, so it was obvious to design a
custom-made shaker adapter and print it with our own in-house 3D printer. This has kept the costs for the
automation of this workflow extremely low. Moreover, the 3D design will be publicly available in our Github repository
, which will make our solution accessible to everyone with access to a 3D printer.
Additionally, we stumbled across serious problems with the calibration of our OT-2 and accessing the shaker with
the pipette. The BioShake D30-T elm is currently not a usual labware defined by Opentrons’, so we had to be
creative and come up with our own labware definition. Opentron is recently rolling out a major update from their
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
decent custom labware. That is why we came up with the idea to use Opentrons’ internal coordinate system and
defining the required 96 Deep Well Plate on the shaker as coordinates. This facilitated accessing the shaker
with the pipette, being as precise as Opentrons’ own labware definitions, but a whole series of problems
followed, as we tried to use Opentrons’ pipette functions to transfer the chemicals. We managed these problems
as well, by defining our own Python functions, telling the pipette how to transfer liquids from and to the
defined shaker. In the end when running the script, one would not be able to tell the difference between the
labware and functions defined by us from the ones defined by Opentrons’.
Putting the pieces together, we were able to translate the manual plasmid purification protocol provided by Nans
Bodet into an Opentrons protocol, being the very first of its kind. We pioneered a workflow for up to six
samples with the p300 Single-Channel Electronic Pipette and a scaled-up version for up to 48 samples with the
p300 8-Channel Electronic Pipette without having to intervene even once. This scalability provides important
flexibility for various kinds of experiments.
In our process of developing and running the protocol we determined some problems on increasing the yield of our
plasmids. There was a large number of parameters that could be varied, changing the final concentration of the
plasmids. For example, we realized that the duration of lysis is paramount for the yield and success of plasmid
purification. Over-lysis will lead to a decrease in plasmid yield, whereas under-lysis will induce clumping of
magnetic beads; thus failing the experiment. After a whole heap of plasmid purifications we managed to identify
the most relevant parameters and improve the protocol in the best way possible.
