Bioscience Technology and Mike Hawes of Dolomite discuss the potential of sorting polymerases using the droplet-based optical polymerase sorting (DrOPS) technique developed in the Chaput lab. The Chaput lab’s synthetic biology research with DrOPS was recently featured in the Nature Communication paper of “A general strategy for expanding polymerase function by droplet microfluidics“.
Developing Novel Polymerases One Drop at a Time
Fri, 09/02/2016 – 10:11am by Mike Hawes, CEO Dolomite
Artificial nucleic acid molecules – such as threose nucleic acids (TNAs) – are attractive to synthetic biologists for a wide range of applications, from catalyzing biological reactions to the storage of data. Benefits of TNAs include their exceptional stability against nuclease degradation and their potential to confer novel biological activities. However, until recently, scientists have been reliant on finding naturally occurring polymerases to do the work of joining these molecules together into long strands, with limited success. Professor John Chaput, formerly of Arizona State University’s Biodesign Institute (now at the University of California, Irvine), said: “Although some natural polymerases are capable of performing this function, efficiency is generally low. Screening efforts have identified a family of thermophilic polymerases from Archaea that work significantly better than most others, and so our research – published April 2016 in Nature Communications – has focused on using directed evolution techniques to improve the capacity of these enzymes to polymerize synthetic nucleic acids.”
The multi-disciplinary team of molecular biologists, engineers and chemists at the Biodesign Institute has developed a novel microfluidics-based method to allow high throughput screening of candidate enzymes. The DrOPs (droplet-based optical polymerase sorting) technique uses an optical sensor to monitor polymerase activity inside uniform water-in-oil-in-water microdroplets. The major benefits of this approach are the ability to screen individual cells or droplets in a high throughput format and the very low reaction volumes required. Chaput said: “Economy of scale is hugely important to the success of molecular evolution techniques and, as all of the artificial nucleic acid substrates need to be chemically synthesized, miniaturization is the only way to make screening economically viable.”
Dr. Andrew Larsen, a former Ph.D. student in Professor Chaput’s lab, said: “Microfluidics was chosen for generating the emulsions because of the uniformity and reproducibility of the droplets. There were already several papers highlighting the success of this approach for the directed evolution of other enzymes, and the ability to easily generate millions of droplets of the same size daily was very attractive; reproducibility is a key component of any screening approach. We initially considered trying to develop our own microfluidics system and chips, before discovering that a commercially available option (Dolomite, UK) would be perfectly suited to our needs. The advantage of using commercially produced chips is that they offer enhanced reproducibility and increased flexibility, with a number of different configurations available to suit a variety of applications.”
Although water-in-oil droplets are sufficient to provide the physical barrier required to create a suitable size reaction environment, an organic carrier is not compatible with commercially available fluorescence-activated cell sorter (FACS) technologies. The Biodesign Institute overcame this by performing a second compartmentalization step to create ‘double emulsion’ water-in-oil-in-water droplets. Larsen said: “The ability to use FACS is important, as this is such a mature and reliable technology. Combined with the very uniform size of the droplets, this allows very efficient sorting and allows us to directly compare the polymerase activities of each mutant. Although we initially used a series of chips to form the double emulsions – and this worked very well – there is now a Dolomite chip available specifically for this application, further simplifying the microfluidic set-up.”
Chaput said: “The quality of the microfluidic chip, and therefore the reproducibility of droplet formation, was really important to us, but it also had to be cost effective. You essentially want ‘plug and play’ technology with a certain level of reliability, but without breaking the bank. If you can buy high quality commercial chips that suit your needs, it’s so much easier to move forward. It also means that other laboratories can potentially benefit from this work and set up their own microfluidics platforms, which will hopefully lower the ‘energy barrier’ to working in this field.”