The overall goal of this DNA assembly protocol is to enable the repeatable scalable automated high-throughput production of DNA devices. This work addresses the need for robust and user-friendly software workflow that can generate pipetting instructions for a liquid handler based on high-level DNA device designs. The main advantage of this technique is that the automated reaction preparation is repeatable and scalable and requires as little as 4%of hands-on time compared to manual preparation.

Visual demonstration of this method is critical because the automation steps can be difficult to learn, and are a combination of experimental and computational methods. A software tool that can automate script generation was developed for this study. Using any web browser, navigate to MoCloAssembly.

com and upload GenBank files for all DNA parts that will be included in the combinatorial DNA device design. Once all the files have been uploaded, select the desired DNA parts. Collections of parts as well as individual parts can be selected.

Drag the parts onto the blank canvas. Order DNA parts such that the five prime and three prime overhangs for each part match. Place part types in the intended final order of DNA parts.

Click assemble on the bottom right of the page. Note that the software tool will only generate valid buildable assemblies based on the four base-pair overhangs that flank each part upon digestion with the BSA1 enzyme. Navigate to the Plans tab and download the files generated by the tool.

These files will include human readable plate maps for the scientists to prepare DNA samples as well as reagents necessary for the reactions, a pick list for the liquid handler and fully-annotated GenBank files for all DNA devices to be assembled. Prior to starting the modular DNA assembly, prepare plasmid DNA as described in the text protocol. The single most critical step of this procedure is correct population of the setup plate and reagent plate using the plate maps generated by the tool.

Following the plate map of the PDF file generated by the assembly tool, place the indicated volume of each diluted DNA part into the appropriate well on a full-skirted 96-well PCR plate. Hold this setup plate on ice until needed. On ice, prepare the reaction master mix with the following components.

For each 20 microliters of reaction, add two microliters of 0X T4 DNA ligase buffer, 0.5 microliters of T4 DNA ligase and one microliter of BSA1 enzyme. Following the plate map for the reagent plate in the generated PDF, distribute the enzyme master mix into the appropriate wells of a new full-skirted 96-well PCR plate. Keep this reagent plate on ice or on a 96-well cold block.

To begin this procedure, place the setup plate and the reagent plate on the deck of the liquid handler. Include an empty full-skirted 96-well PCR plate. The empty plate will be the output plate where the reactions are assembled.

Prepare the liquid handler control software by creating instances of each sample and reagent plate prepared making sure to name them exactly as they appear on the plate maps generated by MoCloAssembly. com including a trough of clean deionized water labeled reservoir. Using the Worklist command in the control software, load the GWL file generated by our software tool followed by another Worklist command which will execute the GWL file loaded in the first command.

Execute the script using the controller software's Run command. After the robotic liquid handler has completed the execution of the script, remove all plates from the liquid handler deck. Save the remaining DNA by sealing the setup plate with aluminum sealing film and storing at minus 20 degrees Celsius.

Seal the output plate with adhesive film, place in a thermocycler or heat block and run with the following cycle parameters. 37 degrees Celsius for two hours, 50 degrees Celsius for five minutes, 80 degrees Celsius for 10 minutes and hold at four degrees Celsius. To maintain sterility, this procedure should be performed near an open flame.

Thaw the requisite number of competent E.coli cell aliquots needed on ice. While the cells are thawing, prepare LB agar plates containing the appropriate antibiotic. Make a master mix containing IPTG and X-GAL.

Pipette 100 microliters of the master mix onto the surface of each plate, and use glass beads to coat the plate evenly. Incubate the plates at 37 degrees Celsius for at least 15 minutes before plating the bacteria. Prepare the transformation plate by aliquoting 10 microliters of competent cells for each reaction into a new 96-well PCR plate on ice.

Add one to three microliters of each reaction from the output plate to the corresponding well in the transformation plate and incubate on ice for five minutes. Seal the transformation plate with adhesive film and heatshock in a thermocycler at 42 degrees Celsius for 30 seconds. Immediately place the plate on ice for two minutes.

To the same wells of the transformation plate, add 150 microliters of SOC media, seal with an adhesive seal, and incubate at 37 degrees Celsius with shaking at 900 RPM for one hour. After one hour, plate the full content of each well of the transformation plate on the LB agar plates prepared earlier. Use glass beads to evenly coat the surface of the plate.

Incubate the plates at 37 degrees Celsius overnight. On the day following transformation, prepare one or several 96-well deep-well culture blocks with 1.5 milliliters of LB broth per well. Using a sterile toothpick, and working near an open flame, pick single white colonies from each LB agar plate of transformed reactions and inoculate the deep-well culture block.

Seal the culture block with a gas permeable seal and incubate at 37 degrees Celsius with shaking at 900 RPM overnight. Subsequently, isolate plasma DNA from bacteria cultures using any commercially available kit, and submit for Sanger sequencing to verify the clones. Reaction assembly times were compared across all three modalities.

Manual assembly took two hours and 10 minutes, all of which was hands-on time. The liquid handler took a similar amount of time to execute the pipetting commands, however only five minutes of that time was hands-on. The acoustic dispenser took significantly less time to execute liquid transfers, and with minimal hands-on time.

This graph shows the cost per reaction for each assembly method. This cost includes the price of enzymes and pipette tips used. Correct sequences were obtained for 95%of the 96 manual and liquid handler sets.

Only 12 of the full 96 assemblies were transformed from the acoustic dispenser prepared samples and 83%were correct. Two reactions failed to yield any white colonies likely due to insufficient mixing of DNA and enzyme master mix droplets in the output plate. A comparison of cloning reaction efficiencies measured by the ratio of white colonies to total number of colonies shows comparable percentages for manually assembled and liquid handler assembled reactions.

Using our tool, researchers can leverage liquid handling robots to construct combinatorial DNA libraries. This method is repeatable, scalable, saves valuable researcher time and reduces the likelihood of pipetting errors that arise from humans performing repetitive complex pipetting tasks. After watching this video, you should know how to generate combinatorial assemblies for modular cloning reactions using our online tool, and how to execute two generated pipetting scripts on a liquid handling robot.

We envision automated DNA assembly with liquid handling robots as just one step in future synthetic biology laboratories. As design complexity increases, we expect approaches like this to be increasingly prevalent and are excited to have represented this first step in this process.

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