Design : Simulate : Execute : Align

High throughput, robust Type IIS DNA assembly workflows, automated by Synthace.

 

Multi step Molecular Biology workflows automated by Antha

The automated Antha Type IIS construct assembly method assembles multiple DNA parts directly from clonal input vectors in a one-pot, room temperature reaction. DNA parts require no special preparation such as digestion, gel purification or PCR, increasing efficiency, fidelity and robustness. Further, the workflow automates transformation, plating out, and setting up of colony PCR.

Upon upload of a simple design file specifying the parts and range of the desired constructs, Antha defines all the required pipetting steps to carry out the assemblies according to the design. These pipetting instructions are sent directly to liquid handling robots that carry out the assembly.

This ensures that complex DNA assemblies are run exactly to protocol, and frees the biologists to think about what constructs they want to test, not how to make them. The use of automation reduces the amount of time at the bench, and dramatically increases the number of assemblies that can be performed robustly.

 
 

DESIGN: The drag and drop user interface allows the researcher to rapidly compose complex workflows.

EXECUTE: Common lab hardware comes pre loaded allowing for quick execution on liquid handling robots.

SIMULATE: Representation of all pipetting actions (blue lines) to make 24 assemblies (purple dots).

ALIGN: Sequence data can also be aligned in the platform.

 
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Higher throughput and increased process robustness

 
 

The Antha Type IIS method assembles DNA parts directly from clonal vectors in a one-pot reaction, at room temperature. This makes it ideal for fully automated execution on simple liquid handling robots.

Antha Type IIS was developed by Synthace through an application of a Design of Experiments (DOE)-based optimisation to Golden Gate Assembly.

This yielded a process that gives colonies 100% of the time for 5-part assemblies, where >95% of those colonies contain the correct construct.

We have tested the optimised automated protocol under a range of conditions and have found it to be robust to variations in environmental conditions and input materials. This robustness has been shown with up to 5-part assemblies, and multiple Type IIS restriction enzymes.

 Antha Type IIS Assembly vs Traditional Cloning

Antha Type IIS Assembly vs Traditional Cloning

 
 
 By conducting a DoE, a substantially simpler and more robust set of conditions were identified (Green box, experiment 2) compared to the previously used Golden Gate protocol (Experiment 1) . These conditions are now known as Antha Type IIS. In the diagrams below, the yellow bars represecent colonies containing the wrong construct.

By conducting a DoE, a substantially simpler and more robust set of conditions were identified (Green box, experiment 2) compared to the previously used Golden Gate protocol (Experiment 1) . These conditions are now known as Antha Type IIS. In the diagrams below, the yellow bars represecent colonies containing the wrong construct.

 
 

Case Study: 100% success rates with Automated Antibody Assembly

Our client, a leading multinational pharmaceutical company, sought to optimise their combinatorial antibody construct assembly and thus looked to deploy Computer Aided Biology by Synthace into their existing workflow. Multiple heavy and light chain constructs were assembled combinatorially, using 3 or 4 components in each reaction with digitally controlled automation, achieving an assembly success rate of 100%.

To undertake this workflow, the design file was simply dragged and dropped into Synthace's platform. The software then seamlessly validated the assembly in silico and detailed the deck and plate layout necessary to set up the experiment. The Gilson PIPETMAX® subsequently undertook the liquid handling steps under the control of the software, saving hours of laborious and error-prone manual pipetting.

Overall assembly timelines were reduced by a third compared to optimised conventional cloning protocols. Because the process was automated and controlled digitally, reproducing experiments was made easy for the user, as was tracking sample provenance during experiments.

Using Synthace's platform and the Gilson PIPETMAX® the antibody construct assembly experiment was designed, simulated, set up and run quickly and precisely. 

All reactions produced colonies, 97% of which contained the clonally correct construct. The three results for each construct are increasing plating volumes.

 
 
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Ready to Revolutionise your Workflow?

The example given here illustrates a proof of concept case for integrating Computer Aided Biology into your DNA Assembly workflows. With applications rapidly coming on-line for advanced bio processing solutions, construct assembly, assay development, and drivers for more pieces of lab hardware the opportunities for transforming your lab’s productivity are endless.

 

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