QUIKCHANGE LIGHTNING ON TRANSCRIPTIC

Ben Miles on 11/4/16 6:04 PM

Part of growing Transcriptic means making industry leading protocols and reagents accessible to our users. For this reason I’m happy to announce that on November 4 2016 Agilent Technologies’ QuikChange Lightning, site-directed mutagenesis kit will be available in the Transcriptic protocol browser.

Earlier in the year, for the first time ever Transcriptic was used in a peer-reviewed study for the generation of a large number of mutants. This enabled the team from UC Davis to explore a parametric space they had previously not had access to. With this in mind we thought enabling the exploration of protein sequence-space was a great application of a programmable lab, as it marries protein engineering computational techniques with wet-lab experimentation.

We approached Agilent who were highly responsive in pursuing the implementation of their QuikChange products on Transcriptic. The first of which that we decided to tackle was QuikChange Lightning for single site-directed mutagenesis.

QuikChange is a very popular suite of kits that provide a highly efficient non-PCR method for reliable site-directed mutagenesis. The QuikChange kits make use of a linear amplification strategy with only the parental strand serving as the DNA template. The kits also feature highly efficient proteins for mutant generation and reaction clean up, which all lead up to a robust and simple user experience.

Schematic cartoon of the QuikChange mutagenesis process.

Schematic cartoon of the QuikChange mutagenesis process.

The implementation on Transcriptic is designed to make it exceptionally easy to generate anywhere from single mutants up to large numbers with minimal hands-on time from the user. A user simply launches the protocol, supplies a .csv list of mutagenic oligonucleotides and the source DNA (typically a plasmid) to mutate.

Once the run has been submitted, the Transcriptic platform takes care of ordering the oligonucleotides, performing the QuikChange reaction, transforming competent bacteria, picking colonies and finally growing the picked colonies in deep well liquid cultures.

A section of the resulting plate from transforming competent bacteria with the QuikChange reaction products. Colonies circled in red were picked by the platform to be grown in liquid culture.

A section of the resulting plate from transforming competent bacteria with the QuikChange reaction products. Colonies circled in red were picked by the platform to be grown in liquid culture.

When implementing partner reagents we have to ensure we can achieve the same data quality as our users are typically used to back at the bench. For QuikChange we replicated the performance of the kit by attempting a two adjacent base mutation from CA to GG. The protocol produced a large number of colonies and upon screening with sanger sequencing 75% of screened colonies were positive for the mutation.

a) Top: Sequence from the source DNA. Below: Sequences from 4 picked colonies. 3 out of the 4 screened colonies were found to be positive for the target mutation. b) Clean sequences traces shown for 3 colonies.

Top: Sequence from the source DNA. Below: Sequences from 4 picked colonies. 3 out of the 4 screened colonies were found to be positive for the target mutation. b) Clean sequences traces shown for 3 colonies.

Over the past 4 weeks, we’ve had the pleasure to talk about QuikChange lightning on Transcriptic at SynBioBeta San Francisco 2016, LRIG Boston 2016 and at iGEM 2016. All fantastic venues for sharing our work and with some very excited people in attendance.

We hope you’ll see how easy it is to start exploring a vast protein sequence-space with Transcriptic and QuikChange Lightning.