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Synthesis of Syn61 genome

With and only with the expanded capacity to handle DNA at 100-kb scale enabled by REXER, the synthesis of a multi-mb bacterial genome becomes feasible. Using REXER and GENESIS, I led the effort and collaboration in the design and de novo synthesis of a fully functional 4-mb recoded genome (named Syn61) in E. coli 3(Fig. 1e).

Kaihang Wang syn61

Fig. 1. REXER and GENESIS enable de novo genome synthesis in E. coli.

In the redesigned Syn61 genome, three out of 64 codons are targeted and completed removed by replacing the target codons (TCG, TCA for Serine, and TAG for Stop) with their synonymous codons throughout all of their occurrences across the entire genome3. Due to the abundance of the three target codons (18,218 designed replacements)3, genome editing to replace them one by one is not realistic, and de novo genome synthesis is the only viable option. The 4-mb Syn61 genome is divided into 38 fragments, each around 100-kb in size3. The entire wildtype E. coli genome is replaced with the Syn61 recoded genome in 38 100-kb REXER steps following the GENESIS strategy3. Modular assemblies were used to speed up the process. With each REXER steps taking one to two weeks to complete, the entire synthesis of the Syn61 genome took more than two years with a joint team of around 20 scientists working full time and a cost of multi-million USD in DNA synthesis, assembly, and validation.

Fredens, J. et al. Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514–518 (2019).