Auto-DNA homologous recombination at a fraction of the cost: Identification of an enzyme cocktail that costs a few thousand times less than commercial reagents by Kyoto University

Recent years have seen the commercial availability of excellent kits that allow two DNA fragments to be joined efficiently by homologous recombination reactions, as long as the overlapping region at the end of the linear DNA fragment is approximately 20 bases long. However, the kits are expensive because they require a variety of enzymes and contain difficult ingredients to prepare, putting pressure on laboratory budgets in exchange for convenience.

Alexander Y. Liu, Hiroto Koga, and Chihiro Goya (all members of iGEM Kyoto, an undergraduate research team at Kyoto University), in collaboration with Makoto Kitabatake (Assistant Professor at the Institute for Life and Medical Sciences, Kyoto University), have developed an enzyme cocktail that enables homologous recombination at a cost several thousand folds less than that of commercial reagents. The study has been published in Genes to Cells.

iGEM, an annual international conference held in Boston, is dedicated to synthetic biology education. Teams of undergraduates from more than 400 universities worldwide compete at the conference by presenting their genetically modified organisms. To enter the competition, iGEM Kyoto raises its own funds, recruits its own staff, and rents a laboratory during the summer holidays for students to carry out experiments based on their own ideas. The iGEM Kyoto team has always suffered from a lack of funding, just like other university teams. However, genetic recombination experiments require the purchase of expensive homologous recombination reaction kits.

Under these circumstances, several members of the iGEM Kyoto team declared, "Let's develop a new reaction solution that can be made without money or experimental equipment." At the time, there was already a published paper reporting that E. coli bacteria could be cultured and treated with a specific surfactant, and the extract obtained from the culture (SLiCE) could be used to perform the homologous recombination reaction. SLiCE was ordered and tested, and its DNA cloning ability was efficient. However, the principle behind the reaction was unknown because SLiCE was a mixture of different E. coli proteins. The team knew that understanding the underlying principle of SLiCE might make it easier to prepare an efficient reaction mixture.

The team reviewed previously known genes of the homologous recombination pathway in E. coli to generate a list of SLiCE drug candidates. Finally, E. coli strains with gene disruptions for seven genes were obtained. When these strains were cultured to produce SLiCE and the products were compared for homologous recombination activity, the extract produced by the strain lacking the gene coding for the enzyme exonuclease III (ExoIII) had no activity.

When homologous recombination was performed with a diluted solution of ExoIII, which was commercially available, it was confirmed that only a minimal amount of the enzyme was enough to obtain sufficient activity.

Based on the known properties of this enzyme, the principle behind the recombination reaction is thought to be as follows: the overlapping sequence of 20 bases at the end of DNA fragments is digested by ExoIII in the 3ʹ to 5ʹ direction, exposing the single-stranded DNA fragments with 5ʹ protruding ends.

ExoIII is unable to digest single-stranded DNA fragments with 3ʹ protruding ends. However, the team observed that this weakness disappears when ExoIII is mixed with exonuclease T (ExoT). Mixing two enzymes could reproduce the full activity of SLiCE, which dissolves many proteins.

The ExoIII reagent responsible for the main enzymatic activity was purchased commercially, but by diluting and using it, the 10,000 yen reagent can be used for 800,000 recombination reactions. By comparison, the commercially available kit costs approximately 1,500 yen per reaction.

Kitabatake noted, "I have struggled with genetic recombination experiments since I was a student. After I became a researcher, convenient kits became available, which made operations easier. However, I have difficulty securing the budget to purchase them. I am delighted that the efforts of these talented and highly motivated students have led to this achievement. I am encouraged that there are many outstanding young people in Japan. I hope those who can afford it will support the activities of iGEM Kyoto."