Imagine its another iGEM day but instead of doing nightshifts you just press a button and your dozens of transformations are processed automatically. Imagine its another iGEM day but instead of plating out mountains of plates you just press a button and plating is done by its own. Imagine its another iGEM day but instead of coming to work early you receive a notification on your phone that all your colonies were already picked and your overnight cultures are currently going through a plasmid purification.
Sounds like another weird dream from a crazy iGEM team, doesn’t it?
Imagine how much time for designing and testing new constructs you would have !
We want to contribute to this dream for the sake of the iGEM community. That's why we started this huge task with the goal to fully automate the processes of building genetic constructs for this year’s iGEM project.
We asked ourselves, which protocols need to be automated the most direly ? We started with small things, writing protocols for pipetting, PCR reactions, eventually combining parts in a one-pot Golden Gate reaction. To make it as user friendly as possible wrote protocols that can be adjusted by a graphical user interface (GUI) (link). We upscaled these protocols to a level at which a single human person would never be able to keep up the pace without making mistakes. Just think about setting up 96 golden reactions with 8 parts each, including buffer and enzymes. This would result in 1056 single pipetting steps for a single 96 well plate!
Continuing down this path we proceeded by automating the transformation protocol using chemically competent cells and the Opentrons’ temperature module. An issue we encountered was the fast transition of the cells from low temperature to 42°C. We implemented the feedback of Keoni Gandall from Drew Endy’s lab in Stanford to pipet the cells rapidly from the cooling block to the temperatur module. This should yield in the highest transformation efficiency possible. We can now transform E. coli in a 96 well format at the same time.
The next milestone on our way was to go from a full 96 well plate to colonies on an agar plate. We had to become a little bit more creative with that: plating every transformation on a single plate was not a valid option as it would limit our scale pretty quickly. Additionally it is up to date impossible to streak out liquids on an agar plate with the Opentrons, which is the manual way that transformations are spread. We decided to follow the advice of Keoni to do 4 dilutions for every transformation and plate all of them on 4 SBS one-well agar plates by spotting 1 µl in an 12x8 array. The higher the dilutions, the fewer cells are spotted per drop, eventually resulting in single colonies.
After having single colonies on our SBS plates, we also implemented colony picking and plasmid purification to finish the whole construct building workflow.
All these protocols for PCR reaction, Golden Gate assembly, transformation and plating are available with detailed documentation on Github, ready to be implemented by other iGEMs team that share our dream of the “lights-out-laboratory”.
We know that we are not saving the world with automating single steps in cloning processes, but we think that these are some essential steps towards a future in which every iGEM team with an Opentrons robot can go to the next level of automation thanks to all our hardware and software solutions. Our vision is to free up their time to focus on designing even more awesome projects, achieving even greater results, dreaming even bigger dreams.