DEVELOPMENT OF BACILLUS CHASSIS AS VECTORS FOR LONG-TERM DNA DATA STORAGE

DNA data storage technology is rapidly evolving to provide long-term stability, compared to conventional hard disk storage. This study takes inspiration from archaeological studies showing that stable DNA samples can be recovered from fossil and microbial samples that have been preserved in permafrost for millions of years. The recent advancement of DNA technology further encourages the developmental trends of DNA storage research, where DNA coding for data storage purposes is supported by its dense capacity, durable stability, and easy transportation. DNA further provides other potential advantages compared to conventional data storage, where it requires low energy consumption and is able to self-replicate, facilitating the ability to withstand the trial of time. However, the technology is currently limited by commercial issues such as the expensive cost of DNA writing and reading for large data sets, despite the decreasing cost of synthesizing and sequencing DNA.

While DNA is considered stable under normal conditions, the stability of the DNA constructs can be compromised by various environmental factors, thus prompting the need for a protective DNA carrier. Currently, various efforts to encapsulate DNA within an inorganic matrix have been employed to extend the shelf life of the DNA. The alternative is to use a living organism to harbor the DNA sequence, where cells can duplicate DNA in the chromosomal or plasmid form with error-correcting capabilities. Thus, bacteria can be considered a suitable chassis, given the rapid growth rate and easy genetic manipulation. In evaluating the various bacterial strains, Bacillus is considered to be a suitable chassis that form spores to survive under extreme conditions while having limited resistance to various antibiotics. The microbial sporulated state enables the microbe to survive in the state of dormancy for an extended time while providing good heat, radiation, and chemical resistance, enabling the stored data to be retained for a longer duration.

Based on the information mentioned above, this thesis reports a method using Bacillus spore integrated with read-controllable artificially assembled bacterial chromosome to facilitate the functional data coding and reading. Additionally, the sporulated state further confers protection to the stored DNA under extreme environment, where these optimizations are suited for long-term data storage applications.

DNA 数据存储技术正在迅速发展,与传统硬盘存储技术相比其长期存储的稳定性更好｡许多考古研究表明,DNA 样本能够在永久冻土层的化石和微生物样本中稳定保存数百万年并能通过现代技术手段回收并恢复,这成为DNA数据存储研究的基石｡如今,DNA生物技术的不断发展进一步推动了 DNA 存储研究的发展｡越来越多的相关研究发现,DNA 存储技术具有存储密度高､稳定性好和易于运输的特性｡利用磁盘作为媒介的传统数据存储技术还存在耗能高和占据空间大的缺点,而DNA存储技术 不仅具有能耗低的潜在优势,并且能够自我复制,可降低其在长时间存储时的数据丢失风险｡现今合成和测序DNA 的成本一直保持逐年下降的趋势,但该技术目前仍受到大型数据DNA 写入和读取成本高昂等商业问题的限制｡

DNA 在普通条件下被认为是一种稳定的生物大分子,但其稳定性可能会受到各种环境因素的影响,因此开发保护性载体是DNA存储技术中重要的一环｡目前,许多研究尝试将 DNA 封装在各种无机基质中以延长 DNA 的存储时间｡也有一些研究使用活的有机体来保存 DNA 序列,比如细菌可以复制具有纠错能力的染色体或质粒形式的 DNA｡考虑到细菌具有快速的生长速度和易于遗传操作的特性,其可以被认为是一种合适的DNA存储载体｡经过评估各种细菌菌株,芽孢杆菌可形成孢子以在极端条件下生存,并只具有有限的抗生素耐药性,是一种出色的工程菌载体｡孢子化状态的芽孢杆菌能够长时间存活,同时具有良好的耐热性､耐辐射性､耐氧化性和耐化学药物的特性,可使存储的数据能够长时间稳定保存｡

根据上述资料,本论文在此报告了一种新的研究方法,我们利用芽孢杆菌孢子结合可控制数据读取的人工组装细菌染色体,以实现极端环境下的数据编码､读取和保护,此方法可为DNA数据存储领域提供一种可行的优化技术手段｡

[1] RYDNING D R-J G-J, REINSEL J, GANTZ J. The digitization of the world from edge to core[J]. Framingham: International Data Corporation, 2018: 16.
[2] ZHIRNOV V, ZADEGAN R M, SANDHU G S, et al. Nucleic acid memory[J]. Nature Materials, 2016, 15(4): 366-370.
[3] DE SILVA P Y, GANEGODA G U. New Trends of Digital Data Storage in DNA[J]. BioMed Research International, 2016, 2016: 8072463.
[4] CEZE L, NIVALA J, STRAUSS K. Molecular digital data storage using DNA[J]. Nature Reviews Genetics, 2019, 20(8): 456-466.
[5] ORGANICK L, ANG S D, CHEN Y-J, et al. Random access in large-scale DNA data storage[J]. Nature Biotechnology, 2018, 36(3): 242-248.
[6] GRASS R N, HECKEL R, PUDDU M, et al. Robust Chemical Preservation of Digital Information on DNA in Silica with Error-Correcting Codes[J]. Angewandte Chemie International Edition, 2015, 54(8): 2552-2555.
[7] CLUZEL P, LEBRUN A, HELLER C, et al. DNA: An Extensible Molecule[J]. Science, 1996, 271(5250): 792-794.
[8] BUSTAMANTE C, SMITH S B, LIPHARDT J, et al. Single-molecule studies of DNA mechanics[J]. Current Opinion in Structural Biology, 2000, 10(3): 279-285.
[9] YAKOVCHUK P, PROTOZANOVA E, FRANK-KAMENETSKII M D. Base-stacking and base-pairing contributions into thermal stability of the DNA double helix[J]. Nucleic Acids Research, 2006, 34(2): 564-574.
[10] AND T A K, BEBENEK K. DNA Replication Fidelity[J]. Annual Review of Biochemistry, 2000, 69(1): 497-529.
[11] BORNHOLT J, LOPEZ R, CARMEAN D M, et al. A DNA-Based Archival Storage System, July[C]. Atlanta, Georgia, USA: Association for Computing Machinery, 2016: 637–649.
[12] MEISER L C, NGUYEN B H, CHEN Y-J, et al. Synthetic DNA applications in information technology[J]. Nature Communications, 2022, 13(1): 352.
[13] WANG Y, HE L, JIANG S, et al. Failure Prediction of Hard Disk Drives Based on Adaptive Rao–Blackwellized Particle Filter Error Tracking Method[J]. IEEE Transactions on Industrial Informatics, 2021, 17(2): 913-921.
[14] RUTTEN M G T A, VAANDRAGER F W, ELEMANS J A A W, et al. Encoding information into polymers[J]. Nature Reviews Chemistry, 2018, 2(11): 365-381.
[15] ALLENTOFT M E, COLLINS M, HARKER D, et al. The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils[J]. Proceedings of the Royal Society B: Biological Sciences, 2012, 279(1748): 4724-4733.
[16] NEIMAN M. On the molecular memory systems and the directed mutations[J]. Radiotekhnika, 1965, 6: 1-8.
[17] CLELLAND C T, RISCA V, BANCROFT C. Hiding messages in DNA microdots[J]. Nature, 1999, 399(6736): 533-534.
[18] BANCROFT C, BOWLER T, BLOOM B, et al. Long-Term Storage of Information in DNA[J]. Science, 2001, 293(5536): 1763-1765.
[19] SANGER F. SEQUENCES, SEQUENCES, AND SEQUENCES[J]. Annual Review of Biochemistry, 1988, 57(1): 1-29.
[20] SANGER F, NICKLEN S, COULSON A R. DNA sequencing with chain-terminating inhibitors[J]. Proceedings of the National Academy of Sciences, 1977, 74(12): 5463-5467.
[21] STADEN R. A strategy of DNA sequencing employing computer programs[J]. Nucleic Acids Research, 1979, 6(7): 2601-2610.
[22] HU T, CHITNIS N, MONOS D, et al. Next-generation sequencing technologies: An overview[J]. Human Immunology, 2021, 82(11): 801-811.
[23] MARDIS E R. Next-Generation Sequencing Platforms[J]. Annual Review of Analytical Chemistry, 2013, 6(1): 287-303.
[24] LU H, GIORDANO F, NING Z. Oxford Nanopore MinION Sequencing and Genome Assembly[J]. Genomics, Proteomics & Bioinformatics, 2016, 14(5): 265-279.
[25] MANI I. Recent development in DNA synthesis technology[J]. New Frontiers and Applications of Synthetic Biology, 2022.
[26] KOSURI S, CHURCH G M. Large-scale de novo DNA synthesis: technologies and applications[J]. Nature Methods, 2014, 11(5): 499-507.
[27] KA. W. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP)[EB/OL] 2022/03/10]. www.genome.gov/sequencingcostsdata.
[28] DONG Y, SUN F, PING Z, et al. DNA storage: research landscape and future prospects[J]. National Science Review, 2020, 7(6): 1092-1107.
[29] YOO H-B, LIM H, YANG I, et al. Flow cytometric investigation on degradation of macro-DNA by common laboratory manipulations[J]. Journal of Biophysical Chemistry, 2011, 02: 102-111.
[30] MATANGE K, TUCK J M, KEUNG A J. DNA stability: a central design consideration for DNA data storage systems[J]. Nature Communications, 2021, 12(1): 1358.
[31] MIKUTIS G, SCHMID L, STARK W J, et al. Length-dependent DNA degradation kinetic model: Decay compensation in DNA tracer concentration measurements[J]. AIChE Journal, 2019, 65(1): 40-48.
[32] SHAO W, KHIN S, KOPP W C. Characterization of effect of repeated freeze and thaw cycles on stability of genomic DNA using pulsed field gel electrophoresis[J]. Biopreserv Biobank, 2012, 10(1): 4-11.
[33] LIU Y, ZHENG Z, GONG H, et al. DNA preservation in silk[J]. Biomater Sci, 2017, 5(7): 1279-1292.
[34] CHEN W D, KOHLL A X, NGUYEN B H, et al. Combining Data Longevity with High Storage Capacity—Layer-by-Layer DNA Encapsulated in Magnetic Nanoparticles[J]. Advanced Functional Materials, 2019, 29(28): 1901672.
[35] SETLOW P, EICHENBERGER P, DRIKS A. Spore Resistance Properties[J]. Microbiology Spectrum, 2014, 2(5): 2.5.11.
[36] CORTESãO M, FUCHS F M, COMMICHAU F M, et al. Bacillus subtilis Spore Resistance to Simulated Mars Surface Conditions[J]. Front Microbiol, 2019, 10.
[37] GOLDMAN N, BERTONE P, CHEN S, et al. Towards practical, high-capacity, low-maintenance information storage in synthesized DNA[J]. Nature, 2013, 494(7435): 77-80.
[38] LUO Y, KORZA G, DEMARCO A M, et al. Properties of spores of Bacillus subtilis with or without a transposon that decreases spore germination and increases spore wet heat resistance[J]. J Appl Microbiol, 2021, 131(6): 2918-2928.
[39] GOULD G W. History of science – spores[J]. Journal of Applied Microbiology, 2006, 101(3): 507-513.
[40] AMADOR ESPEJO G G, HERNáNDEZ-HERRERO M M, JUAN B, et al. Inactivation of Bacillus spores inoculated in milk by Ultra High Pressure Homogenization[J]. Food Microbiol, 2014, 44: 204-210.
[41] NICHOLSON W L, MUNAKATA N, HORNECK G, et al. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments[J]. Microbiol Mol Biol Rev, 2000, 64(3): 548-572.
[42] SETLOW B, SETLOW P. Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from killing by dry heat[J]. Appl and Environ Microbiol, 1995, 61(7): 2787-2790.
[43] LEE KI S, BUMBACA D, KOSMAN J, et al. Structure of a protein–DNA complex essential for DNA protection in spores of Bacillus species[J]. Proceedings of the National Academy of Sciences, 2008, 105(8): 2806-2811.
[44] MARQUIS R E. Bacterial Spores - resistance, dormancy and water status[M]. // REID D S. The Properties of Water in Foods ISOPOW 6. City: Springer, 1998: 486-504.
[45] PONNURAJ K, ROWLAND S, NESSI C, et al. Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation11Edited by I. A. Wilson[J]. Journal of Molecular Biology, 2000, 300(1): 1-10.
[46] HUGHES R A, ELLINGTON A D. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology[J]. Cold Spring Harb Persp Biol, 2017, 9(1): a023812.
[47] BENNER S A, SISMOUR A M. Synthetic biology[J]. Nat Rev Genetic, 2005, 6(7): 533-543.
[48] SHIPMAN S L, NIVALA J, MACKLIS J D, et al. CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria[J]. Nature, 2017, 547(7663): 345-349.
[49] CHEN W, HAN M, ZHOU J, et al. An artificial chromosome for data storage[J]. National Science Review, 2021, 8(5): 028.
[50] VENTER J C, GIBSON DANIEL G, SMITH HAMILTON O, et al. METHODS FOR IN VITRO JOINING AND COMBINATORIAL ASSEMBLY OF NUCLEIC ACID MOLECULES. EP20090711127[P/OL]. 2009-.https://europepmc.org/article/PAT/EP2255013.
[51] DE KOK S, STANTON L H, SLABY T, et al. Rapid and reliable DNA assembly via ligase cycling reaction[J]. ACS SynBio, 2014, 3(2): 97-106.
[52] HORTON R M, CAI Z, HO S N, et al. Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction[J]. BioTechniques, 2013, 54(3): 129-133.
[53] AFRASIAB I, KEAWSOMPONG S, KONGSAEREE P, et al. Formulation of an Efficient Combinatorial Cellulase Cocktail by Comparative Analysis of Gibson Assembly and NEBuilder HiFi DNA Assembly Modus Operandi[J]. International Journal on Emerging Technologies, 2020, 11: 490-495.
[54] ENGLER C, KANDZIA R, MARILLONNET S. A one pot, one step, precision cloning method with high throughput capability[J]. PLoS One, 2008, 3(11): e3647.
[55] IMLAY J A. Transcription Factors That Defend Bacteria Against Reactive Oxygen Species[J]. Annual Review of Microbiology, 2015, 69(1): 93-108.
[56] NAONO S, GROS F. On the mechanism of transcription of the lambda genome during induction of lysogenic bacteria[J]. Journal of Molecular Biology, 1967, 25(3): 517-536.
[57] YASBIN R E, YOUNG F E. Transduction in Bacillus subtilis by bacteriophage SPP1[J]. J Virology, 1974, 14(6): 1343-1348.
[58] ZHANG X-Z, ZHANG Y H P. Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis[J]. Micro Biotechnology, 2011, 4(1): 98-105.
[59] MCDONALD I R, RILEY P W, SHARP R J, et al. Factors affecting the electroporation of Bacillus subtilis[J]. Journal of Applied Bacteriology, 1995, 79(2): 213-218.
[60] ZHANG Z, DING Z-T, SHU D, et al. Development of an efficient electroporation method for iturin A-producing Bacillus subtilis ZK[J/OL]. IntJ Mol Sci, 2015,16(4): 7334-7351.
[61] KO K S, KIM J-W, KIM J-M, et al. Population Structure of the Bacillus cereus Group as Determined by Sequence Analysis of Six Housekeeping Genes and the plcR Gene[J]. Infection and Immunity, 2004, 72(9): 5253-5261.
[62] SOROKULOVA I B, KRUMNOW A A, PATHIRANA S, et al. Novel methods for storage stability and release of Bacillus spores[J]. Biotechnology Progress, 2008, 24(5): 1147-1153.
[63] WEI Y-H, LEE H-C. Oxidative Stress, Mitochondrial DNA Mutation, and Impairment of Antioxidant Enzymes in Aging[J]. Experimental Biology and Medicine, 2002, 227(9): 671-682.
[64] LEE H-C, WEI Y-H. Oxidative Stress, Mitochondrial DNA Mutation, and Apoptosis in Aging[J]. Experimental Biology and Medicine, 2007, 232(5): 592-606.
[65] PFEIFER G P, YOU Y-H, BESARATINIA A. Mutations induced by ultraviolet light[J]. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2005, 571(1): 19-31.
[66] WITKIN E M. ULTRAVIOLET-INDUCED MUTATION AND DNA REPAIR[J]. Annual Review of Genetics, 1969, 3(1): 525-552.