Vector Construction - the easy way
From complex unique 4-part DNA assemblies to protein expression in 4 days.
During my PhD, I remember spending months PCR amplifying my desired DNA fragments and using traditional restriction enzyme cloning tools to generate my final clone. The process went something like this:
I. amplify target DNA with specific primers containing restriction enzyme sites,
II. gel purification of the amplified fragment,
III. restriction enzyme digest of the vector and PCR fragment separately,
IV. restriction enzyme inactivation using a heat source,
V. ligation of vector and inserts using a range of different ratios of the two,
VI. transformation of the ligation mixture,
VII. colony picking and screening several colonies for the correct clone.
I wouldn’t even dare try to assemble more than one fragment into a vector back then.
Today, to perform an identical task, all I do is:
II. assemble the synthesised DNA using the Synthace typeIIS construct assembly in to the desired entry vector,
III. send two clones for sequencing.
The efficiency gains from Antha are clear and are the direct impact of two major industrial revolution synergies: the advent of synthetic biology and the digital age.
At the beginnings of Synthace, we evaluated a whole host of state-of-the-art cloning technologies generated by the synthetic biology revolution, ranging from: 2ab assembly, biobrick assembly, SLIC, isothermal assembly (Gilson), CPEC, SLiCE, Golden Braid, Golden Gate, etc. We were looking for a cloning method that was: high efficiency, accurate, modular for combinatorial assemblies, scar-free, easy to use, low-cost, one-pot, etc.
The typeIIS construct assembly methodology used in the Golden Braid and Golden Gate technologies stood out the most. However, these processes required temperature cycling, weren’t always one-pot and typically had inefficient protocols. Therefore, at Synthace, we did what we do best, we employed DOE (design of experiments), high dimensional multi-factorial experimentation and on liquid handlers, all driven by Antha. The outcome, a typeIIS construct assembly platform that:
I. generates clones 100% of the time*,
II. 99% of the generated clones are correct*,
III. is stable at room temperatures between 18 and 30°C when run using Antha.
Furthermore, our typeIIS construct assembly has all the desired attributes listed earlier! It is: high efficiency, accurate, modular for combinatorial assemblies, scar free, easy to use, low-cost and one pot.
By implementing Synthace’s assembly we have been able to perform high throughput gene assemblies, generating 256 correct constructs in one go for pipeline project without breaking a sweat. All done by using Synthace's software to control multiple liquid handlers with no human intervention other than setting up the liquid handling deck as instructed by Synthace's software.
Ultimately, by transforming the correct assemblies directly into expression strains, we have been able to go from complex unique 4-part DNA assemblies to protein expression in 4 days using Antha. A task that would have otherwise have taken me months to complete using traditional methods or weeks using the current state of the art – and the best thing, I didn’t even have to use a manual pipette!
*True for up to 4-part assembly, not tested upward of 4 parts.