iVDTv2: Directed Evolution with High-throughputCopyright: BMBF
In established methods in Directed Evolution, it is necessary to generate, cultivate and screen several thousands of mutants, in order to obtain an enzyme with one to two amino acid substitutions. Hereby, 60 – 80 Prozent of the amino acid substitutions, which improve the enzyme properties, cannot be identified. In the iVDTV2 project a method for directed evolution is developed, which enables the generation and screening of millions of mutants in relatively short time.
Established protocols are based on bacteria cultures. Random mutations are introduced into the DNA encoding the information for the enzyme of interest. Subsequently, the bacteria reads the DNA and produces the enzyme (gene expression). Afterwards, the enzyme is tested in a reaction, in which a fluorescent product is produced.
Every mutant is expressed in a separate bacteria culture, in order to test and possibly use them separately. This means that thousands of bacterial cultures have to be cultivated, the enzymes of interest have to be purified in order to perform thousands of test reactions.
The biggest limiting factor is the transformation efficiency, because not every gene can be taken up and expressed.
Within the iVDTv2 project, 107 mutants are being expressed and tested within one hour and within one tube. The required spatial separation is achieved by emulsification of the reaction mixture in an oil phase, aqueous droplets are formed that constitute separated reaction compartments and can be broken-up by the addition of detergent when required. The emulsified reaction mixture contains the mutated DNA, all components needed for cell-free protein synthesis and a fluorogenic substrate as an indicator for the enzymatic activity to be monitored. The DNA is added in such a high dilution that an individual droplet statistically only contains one gene and thus one mutant. Within the droplet coupled transcription-translation and indicator substrate cleavage occur in parallel. Droplets with active and improved variants are sorted and enriched by flow cytometry and finally worked-up. The contained DNA is amplified by PCR and is thus available for a new round of evolution.