Authors: Alan Walker & Davide De Lucrezia
For much of its history, the exponential decline in the cost of DNA has been held as the central metric of progress in Synthetic Biology. Often compared to Moore’s law, it has sparked a wealth of startups and new research.
But cost is an incomplete metric that hides many challenges. That’s because, as many DNA customers understand; reliability and turnaround have become the major limits to discovery. In this piece by Gingko the picture is clearly painted: for nearly all suppliers, turnaround time is highly unpredictable and rarely fast enough to make outsourcing a viable part of the development cycle.
A mature synbio market depends on an efficient supply chain like any other. This means that DNA must not only be cheap but as reliable to source as the chips that sparked the digital revolution. The main reason behind the lack of a dependable source of DNA is that several factors influence the manufacturability of DNA, and some of them are not readily detected when uploading a DNA sequence to a DNA providers website.
For a decade, we’ve been building a reliable DNA pipeline at Doulix to support advanced research. And in this time, we’ve learnt there’s a lot you can do to improve your chances of success. So, to help you unblock your synthesis bottleneck, our resident experts Sota Hirano & Florian Nadler share their advice for faster, more successful DNA outsourcing.
- Careful with complexity
This will be familiar but it is still the most common problem we see. Repeats, secondary structures and high GC content cause an array of problems during synthesis and assembly. Simplifying complexity in the design stage with optimization tools can have a big impact on success and turnaround.
- Check your primers
Make sure to avoid stable secondary structures, self/hetero dimers and other fiddly details like having C or G in the first position on the 3’ end, or that the first 6-10 nucleotides from 3’ to 5’ end are not repeated elsewhere.
- Consider toxicity
Always consider toxicity created by your sequence. It could be from a translated CDS product or a regulatory element but either way, it is a problem. This can be an issue with poorly characterised public parts as an overly strong promoter can mean delays due to trialling multiple cloning methods and screening hundreds of colonies.
- Plan for failure
When working on custom builds, whereby a successful protocol has not yet been proven, we will adapt sequences to allow for contingencies. We’ll add restriction sites to linearize a plasmid as a backbone in case PCR approaches fail; we’ll design overlapping regions to be compatible with multiple assembly methods or design multiple primer pairs to increase resolution for colony screening PCR.
Ensure unwanted gene expression is suppressed in the cloning strain to avoid unnecessary metabolic burden and selection of deleterious mutants. Check your promoters are regulated in the cloning host and ensure proper terminators to prevent accidental read-through.
- Finally, treat it like a relationship
Working in a Synbio laboratory is like a relationship; it needs constant attention. Or else, the moment you get complacent is the moment it all goes wrong. What’s the moral? Whether you’re outsourcing or staying in house, never take your protocols and tools for granted.