Barstow Research Team

Department of Chemistry, Princeton University

Note From Authors

In the coming century, the world faces a crisis of energy, sustainability and climate. In the next 25 years, world energy use is projected to increase from 17 terawatts (TW) to 26 TW. If this energy is supplied by fossil sources, it will pollute the atmosphere with carbon dioxide with consequences for climate, ecology and society for centuries to come. Even if this carbon is sequestered, we are still faced with the peak in production of coal, the most abundant fossil fuel, before 2050.

Biology already gives a first draft solution to the problems of non-polluting energy capture and storage at the scale needed by civilization, channeling ≈ 100 TW by photosynthesis into biomass. Furthermore, biology is self-replicating and constructs itself from abundant elements, lowering deployment costs.

We realized that we needed much better genetic engineering and characterization tools for electroactive microbes, with a whole genome knockout collection being a top priority. While a whole genome knockout collection is one of the most useful tools for gene discovery, the high costs of constructing them mean that they are out of reach for many of the most interesting organisms. Even with the dramatic recent advances in next-generation sequencing, the percentage of genes of unknown function in a genome sequenced today remains approximately the same, 30-40%, as it did a decade ago. This means that many of the genes that could be used to engineer microbes for sustainable energy and green chemistry applications, or that are responsible for the synthesis of novel therapeutic molecules or antibiotic tolerance go undiscovered.

We developed a technology, Knockout Sudoku, that would allow us to rapidly and cheaply construct whole genome knockout collections. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, which is a 30- to 100-fold improvement over prior methods. We now have a unique resource that can teach us how to use these microbes in applications across sustainable energy including the safe use of nuclear power; enabling the widespread use of batteries; and electrosynthesis.