Team:Marburg/Cloning Protocols

C L O N I N G   P R O T O C O L S

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 we wrote protocols that can be adjusted by a graphical user interface (GUI). 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.

G.U.I.D.E - Graphical User Interface for Directed Engineering

A large part of the work that we as biologists do in the laboratory consists of repetitive, monotonous, mundane tasks. Imagine a world in which the scientist is liberated from these experiences and is able to fully concentrate on the important things of their project. Enter the world of automation. We firmly believe that affordable lab automation will fundamentally change the field of biology as we know it today. In order for this change to be a positive one, we consider it indispensable to take greater account of the end-user.

Unfortunately, most of us had the experience that a great deal of software solutions in science are too difficult to use and therefore discourage many potential users from the get go. In order to tackle this problem, this year’s team has given a lot of thought into what makes a good user experience and how to use this to bring our automation efforts to a wider audience. Our project required a variety of Opentrons OT-2 protocols that range from simple dilution series to much more complex Golden Gate assemblies.

To reconcile this diverse set of protocols without overwhelming the user, it requires intuitive software that packages modern features, such as drag and drop support, an easy to use deck overview and intelligently groups features that are more advanced.

Fortunately, these ideas thoughts came around the same time as the invitation to the BioHackathon from iGEM Vilnius, allowing us to put them directly into practice. The Hackathon gave us the unique opportunity to discuss our efforts with other iGEM teams and experts from different disciplines, making it possible to immediately implement the feedback.

We worked on an app were protocols can be integrated simply via drag and drop. After successfully loading the protocol into its library, the user can see not only the labware used, but also the liquids and their correct position on the OT-2 deck. Additionally the applications allows for protocol simulation, advanced code view and the change of variables

After just 3 days our prototype had many of the aforementioned features making the participation a resounding success. Later development allowed us to expand its functionality and smooth out its edges.

The user interface (UI) is written in C# and can interface modified Opentrons protocols that are written in python via json files that define variables such as labware and liquids. In order to make the UI more visually appealing we designed multiple vector graphics of the labware and robot.

We believe that this is a step in the right direction for a more user-friendly automation environment and hope to build upon this experience to expand this project in the upcoming years.