At Cradle, our team is exploring new methods in mutagenesis, aiming to efficiently and cost-effectively create gene variants that produce the proteins we need. Protein engineering is at the heart of our work—it's essential for developing solutions ranging from eco-friendly laundry detergents to personalized medicines. These innovations are key as we move from a traditional industrial age into a sustainable bioindustrial future.

Understanding the Darwin Assembly Method

One of the methods we extensively researched and experimented with is the Darwin Assembly method, a gene mutagenesis approach, originally developed by Christopher Cozens and Vitor Pinheiro. This method enables scientists to rapidly introduce multiple mutations in a single construct, expanding the design space and increasing the potential for optimizing different protein characteristics simultaneously.

Advantages over other mutagenesis methods

During our research, we found that Darwin Assembly offers several advantages over other mutagenesis methods. It provides full control over the sequence at a cost similar or slightly more than random mutagenesis. Unlike other methods such as de novo gene synthesis, the cost of DNA made using Darwin Assembly doesn't increase with fragment length. Additionally, it is designed to introduce up to 8 mutations in a single construct at once, which is a significant advantage over classical site-directed mutagenesis, the default method chosen by financially limited research groups. Classical site-directed mutagenesis can only introduce mutations at a single site or location in the gene, while Darwin Assembly allows for multiple simultaneous mutations.

Cost-effectiveness and accessibility

The Darwin Assembly method starts with a plasmid containing the wild type sequence (or base / starting sequence). First, a nicking restriction enzyme nicks one strand of the DNA. Then, an exonuclease chews back the nicked strand, leaving a single-stranded plasmid onto which mutagenic oligos (up to at least 8, and likely more) are annealed. A DNA polymerase fills the gaps, a ligase seals the nicks, and the new, mutated strand is then amplified by PCR. Flank sequences used to facilitate the PCR reaction can also be leveraged for Gibson assembly of the construct into any desired vector for protein expression. This method is accessible to virtually any lab, and throughput can be increased using cost-effective instruments like the OpenTrons OT-2 or an I.DOT liquid handler.

Cradle's Experience with Darwin Assembly

While the success rate of going from in silico genes to actual plasmids containing the gene variant is slightly lower for Darwin Assembly compared to other methods, we found the Darwin Assembly method to be promising and spent considerable time experimenting with it. However, we encountered some challenges in making the method consistently robust. We observed rounds of decent build efficiencies (>60% success rates) followed by very low efficiencies (10-20% success rates). In these low efficiency rounds, we noticed a significant amount of template sequence bleeding through instead of our intended variants. Although we believe it's possible to iterate on the method to achieve consistent robustness, we were constrained by time. Our priority was to generate data to validate our models immediately, rather than spending several months optimizing the method. Ultimately, our team decided to collaborate with Twist Bioscience for our DNA needs. This decision was based on several factors, including the range of services and expertise offered by Twist Bioscience, the quality and speed of their deliveries, as well as the potential for a long-term, mutually beneficial partnership.

Sharing Knowledge and Insights

Despite our decision to work with Twist Bioscience, we believe that the knowledge and insights gained from our research and experimentation with the Darwin Assembly method can be valuable to other researchers and organizations in the field of protein engineering. By sharing our experiences, we hope to contribute to the growing body of knowledge surrounding efficient and cost-effective methods for designing, generating, and testing proteins.

As the demand for diverse, optimized proteins continues to grow, it is crucial that research groups have access to methods that allow them to produce exact gene sequences rapidly and cost-effectively. While the Darwin Assembly method may not be the best fit for every organization, it offers a unique combination of control, cost-effectiveness, and accessibility that makes it worth considering for those seeking to advance their protein engineering programs.

Protocol
We have open-sourced our protocol on Briefly.bio.

Original paper
Cozens, C., & Pinheiro, V. B. (2018). Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis. Nucleic acids research, 46(8), e51.

Contact us today to learn how to get early access to Cradle’s protein engineering platform and advance your protein engineering programs.