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.
![Connections between Opentrons, Promega and QInstruments](https://static.igem.org/mediawiki/2019/6/60/T--Marburg--SyntexConnections.png)
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.
![OT-Layout left](https://static.igem.org/mediawiki/2019/e/ea/T--Marburg--SingleChannelSetup.png)
![OT-Layout right](https://static.igem.org/mediawiki/2019/d/df/T--Marburg--8channelSetup.png)