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 discontent, the idea was born to develop a user-friendly software to accelerate the wet-lab workflow.
We 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”.
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.
This 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. TEIL VON CARLOS
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 zu Renes Text). Certainly, we also used Click ‘n’ Clone to create picking lists and showed the compatibility of our software with this robot.
In summary, we developed a user-friendly, easy to use software tool that provides a GUI and allows users to select parts for creating desired LVL1 plasmids. A pipetting protocol can be exported for manual operation in table view or in the shape of a picking list for pipetting robots. We created Click ‘n’ Clone based on the need to reliably assemble high numbers of independent plasmids and we managed to accelerate an everyday wet-lab workflow. We also created an interface to generate picking lists for pipetting robots in an easy and convenient manner.
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<figcaption><b> Figure 2: </b>Pipetting protocol generated by Click 'n' Clone </figcaption> | <figcaption><b> Figure 2: </b>Pipetting protocol generated by Click 'n' Clone </figcaption> | ||
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This 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. <br><br> | This 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. <br><br> | ||
Revision as of 10:00, 13 October 2018
Software Click 'n' Clone