The Institute for Research in Immunology and Cancer (IRIC) at the University of Montreal is using a novel technique to investigate complex biological processes. IRIC is aiming to develop new techniques in biomedicine and drug discovery by combining genomics, bioinformatics, and proteomics with systems and synthetic biology.
Professor Michael Tyers, who is leading the Systems Biology and Synthetic Biology Research Unit, is investigating the mechanisms of cell growth and division to unravel interactions at the genetic and proteomic levels.
The information, thus gained, can be used for re-engineering natural networks and building fully artificial networks in order to perform novel biological functions. Molecular cloning plays a vital role in the group’s research and enables them to develop models of complex biological processes.
When throughput demands increased, automation was looked as an opportunity to considerably streamline this process as well as eliminate unnecessary repetitions and errors. The laboratory team established a fully automated, reliable cloning workflow on a Freedom EVO® workstation.
This system performs customized plating in ANSI/SLAS-format plates, fully automated multi-fragment DNA assembly, colony picking and PCR set-up, and also agar plate preparation, thus enabling the group to increase the throughput of their experiments.
The ability to integrate upstream and downstream tasks with colony picking using a Pickolo™ Colony-Picker (SciRobotics) (Figure 1) offers the laboratory a flexible solution, thus substantially contributing to the success of their projects.
Figure 1. Automated cloning workflow on the Tecan Freedom EVO® workstation.
Material and methods
IRIC’s Freedom EVO® 200 workstation is fitted with a robotic manipulator (RoMa) arm and an eight-channel liquid handling (LiHa) arm. Two chilling/heating devices – EchoTherm™ RIC20XR and RIC20XT (Torrey Pines Scientific) – are employed to pre-warm the agar for plating and water for wash steps, respectively.
The integrated Pickolo™ Colony-Picker includes a high-resolution camera fitted on the LiHa arm, a backlight carrier, and the Pickolo™ software.
The platform is also equipped with a Te-VacS™ vacuum station for DNA extraction and clean-up, a temperature-controlled incubator (MIO™), and two orbital shakers (BioShake® 3000, Q.Instruments) as well as microplate carriers and shelves for additional on-deck storage. A barcode reader fitted at the rear of the workdeck enables performing full tracking of barcoded plates.
Figure 2. Freedom EVO® 200 worktable overview.
Plate preparation and plating procedure
Two different methods were developed to ensure that the complete cloning workflow can be performed using ANSI/SLAS-format plates. These methods allowed plating into either standard 6-well and 12-well culture plates (BD Falcon™, flat bottom with lids) or 8-row polypropylene (PPE) reservoir plates (Seahorse Bioscience Labware, Figure 3).
Figure 3. 8-row PPE reservoir plates were used for an alternative plating strategy.
For both plate types, a single-well reservoir was used to maintain the pre-boiled agar at approximately 70 °C (with the heater set to 100 °C). Then, the agar is transferred to each lane or well by employing multiple aspirate-dispense steps with either three, four or eight tips in parallel to result in agar plates with highly reproducible fill volumes.
Plating into 8-row PPE reservoir plates
After pipetting 100 μl of bacterial suspension into one end of all the lanes, the plate is lifted at the same end using the RoMa arm. This allows the bacterial suspension to spread evenly along the agar surface.
Plating into 6- and 12-well plates
By implementing a customized Freedom EVOware® LiHa firmware command, a spiral-like motion of the tip during pipetting is enabled, as shown in Figure 4. At the time of dispensing, the tips of the LiHa arm move in X- and Y-directions by turns, thus forming a square spiral plating pattern, and ensuring an even spread of the bacterial suspension across each well.
Figure 4. Graphic representation of the spiral tip movement for plating on 6- or 12-well ANSI/SLAS-format plates.
After plating, the culture plates were externally incubated for 8 to 16 hours at 37 °C before colony picking, employing a customized picking profile; the agar surface was identified through conductivity. This was followed by a small, 0.6 mm to-and-fro ‘scratching’ movement of the pipette tip in order to ensure reliable transfer of adequate bacterial material for inoculation.
Four to eight colonies were picked per lane or well, moved to a 96-well PCR plate for PCR confirmation, and copied to a deep-well plate for storage and further analysis. After PCR, agarose gel electrophoresis was used to evaluate positive clones offline.
Results and discussion
Successful preparation of agar plates
Culture plates with fewer air bubbles and precisely controlled fill volume are obtained by implementing an automated agar plate preparation procedure. This ensures high picking accuracy for the Pickolo®.
Two different methods exist for plating out of transformed bacteria into ANSI/SLAS-format culture plates. These methods are a practical alternative to the use of Petri dishes for complete automation of cloning workflows.
The plating procedure described for 8-row reservoir plates is quicker, and offers a higher throughput for many of the lab’s applications. On the other hand, plating into 12- or, especially, 6-well plates in a square spiral pattern achieves more robust single-colony picking from cultures with extremely high cell densities.
The software’s flexibility allowed a customized picking profile to be created, similar to the manual picking movement with a small sideways scratch. This ensures that sufficient material is moved to the PCR plate as well as the deep-well storage plate. Colonies can be reliably collected and samples can be tracked throughout the whole workflow by employing this automated procedure.
The Freedom EVO® platform’s flexibility has enabled the group to close two gaps in their automated cloning workflow: plating of bacteria onto ANSI/SLAS-format culture plates and agar plate preparation. Additionally, the platform’s flexibility has created a reliable, powerful, fully automated cloning solution for synthetic biology research.
We would like to thank Dr Raik Grünberg, Dr Almer van der Sloot, Xingjian Xu and Prof Michael Tyers (IRIC, University of Montreal, Canada) for kindly sharing information and their experiences of setting up the automated workflow on the Tecan Freedom EVO® platform.
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