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.

Connections between Opentrons, Promega and QInstruments
Figure 1 - iGEM team Marburg 2019 is establishing connections between Opentrons, Promega and QInstruments.

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’.

OT-2 left OT-2 right
Figure 2 - Single-Channel pipette, magnetic module and shaker in action while performing the plasmid purification.

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.

OT-Layout left OT-Layout right
Figure 3 - Final setup for the automated plasmid purification workflows in the OT-2. The left picture shows the setup for the single channel workflow, the right picture for the 8-channel workflow. The wells A1 to A10 for the single channel setup contain the chemicals in the following order: resuspension solution, lysis solution, neutralization solution, Magnesil Blue, Magnesil Red, Isopropanol, wash solution, ethanol, ethanol, nuclease-free water.