2020 Volume 60 Issue 5 Pages 284-287
In vitro reconstitution of cellular events is a promising strategy as a bottom-up approach to build an artificial cell in the field of synthetic biology. Also, the reconstituted system sometimes provides a useful biotechnology tool for various fields. We recently reconstituted a whole replication cycle of the Escherichia coli chromosome in vitro as a part of the bottom-up synthetic biology. This replication cycle reaction (RCR) propagates circular DNA exponentially and precisely by autonomous repetition of the cycle in an isothermal reaction. Even genome-sized DNA (1 Mbp) can be propagated as circular DNA in RCR. We also developed a DNA assembly tool. The combination of the DNA assembly and RCR amplification enables cell-free synthesis of artificial large circular DNA, providing a powerful tool for a top-down study of the synthetic biology, in which living cells are modified at a whole genome level.
