Storytelling:

This year’s iGEM 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 (GitHub Link?), 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 (Link to IHP) 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, a massive amount of barriers had to be broken down. 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, which would keep the costs for the automation of this workflow extremely low. Moreover, the 3D design will be publicly available in our GitHub repository (LINK), 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’.

While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in light measurement, evaluated different reporters???, established a measurement method and compared it to a already known FACS measurement method (?).

At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle]. So we had to measure the light conditions in our incubators and while doing this simple task the first part of standardization began. We discovered that nearly every paper? is using different methods to measure their light conditions and that it is a really complex and important procedure. So we got in contact with cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following popup we show different ways of measurement, their (dis-)advantages and different results depending on the measuring instrument.
Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. In literature and while talking with different experts (IHP), we recognized that small deviations of these parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters that lead to the fastest growth speed.
Another aspect was measuring the expression and characterize our part. Different possibilities were discussed and after testing them we decided on two methods in our project (plate reader and FACs). One approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.
The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the difference in measurement methods.
At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973 and make a contribution to the cyano community by establishing essential/fixed standards in measurement.

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.