From the genes contained in our DNA, the proteins within our bodies are born. As researchers progress in their ability to manipulate DNA, they are beginning to design and create synthetic proteins with a wide array of applications, such as new medical treatments, biofuel production, breakdown of pollutants, and biosensors that can detect specific molecules. To create synthetic proteins with new properties, scientists often need to modify DNA sequences or create entirely new ones. They then need to test the resulting genetic variants to see if they will produce a protein with new functions that could be useful for their desired application. Read More
However, introducing and then testing these modifications one at a time is incredibly time consuming, and each modification may have relatively modest effects on the activity of the final protein.
To speed up this process, scientists need techniques that can introduce several modifications at once in particular regions of DNA and produce large sets or combinations of different modifications. These large sets are called libraries. A method to rapidly test the potential of these many genetic variants is also necessary.
Towards this aim, Dr. Ramesh Jha and colleagues at the Los Alamos National Laboratory in New Mexico developed a technique that allows them to rapidly introduce a variety of modifications at each target site, using relatively simple laboratory techniques, such as polymerase chain reaction (PCR).
By using PCR primers with variable bases at certain positions, they can quickly generate gene fragments that carry one or more modifications of interest.
They can then introduce further diversity by fusing many DNA fragments together in different combinations. This approach allows the researchers to create libraries that contain hundreds of thousands to tens of millions of genetic variants.
The next step involves testing and identifying useful combinations of mutations within these libraries. Finding such “variants of interest” involves first introducing the mutated DNA into bacterial cells, in a process called transformation, which will allow the bacteria to express the proteins encoded by the genes.
The genetic variants are then linked to a gene circuit which, based on the performance of the genetic variant in the bacterial cell, produces more or less of an encoded fluorescent protein. This allows variants of interest to be rapidly identified by the brightness of the fluorescing cells, which can then be isolated and collected using a technique called fluorescence-activated cell sorting (FACS).
FACS involves flowing the cells individually and rapidly past a fluorescence detector, which will detect and then divert fluorescent and non-fluorescent cells into separate collection tubes. In this case, the fluorescent cells represent those in which a variant of interest is present. These variants can then be explored further, while non-useful variants can be discarded.
While Dr. Jha’s new approach uses existing and well-known laboratory techniques, it combines them in a unique and previously unreported combination. The resulting methodology bypasses the existing bottleneck in creating and testing genetic mutations. The team’s approach could drastically reduce the cost, time and tedious labor involved in developing new proteins with exciting and useful functions.
