| With the rapid growth of digital information,DNA emerged as a novel potential material for mass data storage,offering the possibility to cheaply solve a great data storage problem.DNA data storage is divided into in vivo and in vitro storage.At present,the storage capacity of in vivo is much smaller than in vitro storage system.In addition,the low-bias amplification of large DNA pool is a challenge in in vitro storage because of many factors(such as GC content and the length of DNA sequences,DNA polymerase)leading to amplification bias.Here,we studied the problem of small storage capacity in vivo and amplification bias of storage in vitro.We first showed a mixed culture of bacterial cells carrying a large DNA pool with encoded digital information and assembled in a high-copy-number plasmid by a redundancy method,and we analyzed the preference of sequences in DNA pool in mixed culture.According to deep bioinformatics analysis,although there was sequence context dependent bias in homologous assembly,the information of large DNA pool remained stable in multiple successive passages of mixed culture.Finally,over ten thousand distinct oligos encompassing 2304 Kbps and encoding 445 KB digital data were stored in cells successfully,which is the largest storage in living cells reported so far.Moreover,we systematically explored the influence of the GC content on the amplification of a pool comprising 11520 distinct strands with moderate GC content in the range of 35-65%.As expected,oligos in the pool were amplified unequally,but contrary to the existing theories,the significantly enriched oligos all had relatively high GC content.Further thermodynamic analysis demonstrated that relatively high value of both GC content and Gibbs free energy(double-G)could improve the replication of specific oligos during biased amplification.In summary,we successfully combined the DNA pool for in vitro storage with the advantages of in vivo storage system to achieved large-scale DNA data storage in vivo,and present a new idea to bridge the gap between in vitro and in vivo storage systems;Moreover,double-G driven replication advantage can be used as a guiding principle for the design of large-scale DNA pools for in vitro storage.Our study can lay the foundation for the development of both in vivo and in vitro storage. |
