The overall goal of our project is to accelerate scientific progress. For this reason we aimed to establish V. natriegens, the fastest growing organism, as a new chassis for synthetic biology. We additionally created the Marburg Collection to facilitate easy and reliable cloning of plasmid constructs. At the moment when we started to assemble large numbers of plasmids for our characterization experiments, we realized that selecting parts from the list of LVL0 plasmids, calculating molar ratios and DNA amounts can be really time consuming. From this inconvenience, the idea was born to develop a user-friendly software to accelerate the wet-lab workflow.
The creation of a cloning softwareWe envisioned a software that takes plasmid sequences and DNA concentrations as input and provides the users with a detailed pipetting protocol that considers the different plasmid sizes and accepts given DNA concentrations. To create a user-friendly input, the optimal software should include a clearly arranged graphical user interface ( GUI ). Additionally we wanted to implement the feature that a user can set the desired ratio between resistance and all other parts, which can be found in many golden-gate protocols. We started to work on a software tool that fulfills all these requirements and termed it “Click ‘n’ Clone”.
To start working with Click ‘n’ Clone, a user just has to generate a batch export of all LVL0 plasmids as fasta files from a cloning program like Geneious. The software automatically determines the size of each plasmid and its name from the batch export. The plasmids are sorted in categories based on the nomenclature. For example, all promoter parts are sorted in one category and all terminator parts are sorted in a different category. All parts are assigned to lists, that are displayed in a graphical user interface. Users can select one part of each category that shall be assembled to a LVL1 plasmid. Additionally, the DNA concentration or molarity of the resistance part can be set by the user and a molar ratio can also be specified.
Now, all the required information for calculating the pipetting protocol are available and a plasmid name can be entered. By pushing the button “calculate” the software reads the user specified values for DNA concentration and molar ratios, calculates the required fmol and ng of each part by considering the size of each individual plasmid. Based on the concentration that is provided by the csv files, the volume that needs to be pipetted is computed.
The result of all calculations is displayed in a table within the GUI . For documentation reasons, this table can be saved and exported as a csv file which can be copied or printed for your lab book.
Bringing Click 'n' Clone to the labThis first version of Click ‘n’ Clone was designed for creating pipetting protocols for manual operation. And already truly helped us in our everyday lab work by shortening the amount of time required for setting up golden-gate reactions.
During april this year we noted that iGEM started a collaboration with Opentrons, a company that develops and sells low-budget pipetting robots. We applied for a free robot, succeeded and finally received Opentrons OT-2 pipetting robot. We wondered if we could utilize this machine in combination with Click ‘n’ Clone to reliably set up a large numbers of golden-gate reactions in a short amount of time. We modified Click ‘n’ Clone in a way that the output is transformed from a table into a picking list that contains information about the source and destination well and the volume that needs to be transferred.
Unfortunately, the OT-2 is not capable of directly importing csv files. To overcome this limitation, we developed an additional Python script to make the picking lists accessible for the robot.
The script is divided in two subscripts, a reading and executing script and an auxiliary script containing the commands for the Opentrons. By dividing the scripts the work was kept easy and clear. Furthermore we kept the possibility to code the workflow for the robot straight forward and the possibility of testing the script.
The auxiliary script contains all the information about the labware, pipettes, tips and other containers set in the robot. It needs for every step a source point, a destination and the volume to be transferred. To save space in the Opentrons the script checks after every pippetting the number of tips remaining. When the first box is empty it gives a message and keeps working with the second tipbox. If this is emptied as well the robot pauses and waits until both boxes are refilled again. This way we could use every kind of well plate and pippetting scheme without worrying running out of tips. We tested the script several times and fine-tuned it until all steps worked flawlessly. When all bugs were fixed the executing script was edited. Basically the important thing here is the path were the auxiliary script and the Click’n’Clone output were located. The rest runs automatic. In the first step the script reads the .csv file and orders the single values in three lists for source, destination and volume. In the next step the auxiliary script is read and every line saved in another list. Finally the lines for the pippetting coordinates are replaced by the read output and the list is saved as a new .py file. This file is ready for upload into the Opentrons. The combination of Click’n’Clone with this automated script made our work extremely fast and easy.
The scripts can be downloaded by clicking on the link below.
We tested this workflow experimentally and realized that a constant volume of each sample is advantageous as the OT-2 shows decreasing accuracy for small volumes. Therefore we diluted all LVL0 plasmids to a uniform concentration and transferred them into a 384 well plate. We created picking lists using Click ‘n’ Clone, imported them into the OT-2 via the Python script and afterwards watched the robot combining LVL0 plasmids. At the end of the day we obtained 50 plasmids that were designed with Click ‘n’ Clone and subsequently pipetted with a robot.
In addition to the OT-2 , we were granted the opportunity to work with the acoustic liquid handler Echo for a short period of time (Link to Labautomation) . Certainly, we also used Click ‘n’ Clone to create picking lists and showed the compatibility of our software with this robot.