WO2013169867A1 - Procédés et compositions pour stockage de données numériques réinscriptibles dans des cellules vivantes - Google Patents

Procédés et compositions pour stockage de données numériques réinscriptibles dans des cellules vivantes Download PDF

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WO2013169867A1
WO2013169867A1 PCT/US2013/040089 US2013040089W WO2013169867A1 WO 2013169867 A1 WO2013169867 A1 WO 2013169867A1 US 2013040089 W US2013040089 W US 2013040089W WO 2013169867 A1 WO2013169867 A1 WO 2013169867A1
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integrase
excisionase
dna
storage system
data storage
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PCT/US2013/040089
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Jerome Cedric BONNET
Pakpoom SUBSOONTORN
Andrew David ENDY
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The Board Of Trustees Of The Leland Stanford Junior University
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    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06N—COMPUTING ARRANGEMENTS BASED ON SPECIFIC COMPUTATIONAL MODELS
    • G06N3/00—Computing arrangements based on biological models
    • G06N3/12—Computing arrangements based on biological models using genetic models
    • G06N3/123—DNA computing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic

Definitions

  • Such epigenetic storage systems can be subject to evolutionary counter selection due to resource burdens placed on the host cell or spontaneous switching due to putatively stochastic fluctuations in cellular processes including gene expression.
  • engineered transmission of DNA molecules could support data exchange between organisms as needed to implement higher-order multicellular behaviors within programmed consortia (Ham TS, Lee SK, Keasling JD, Arkin AP (2008) Design and construction of a double inversion recombination switch for heritable sequential genetic memory. PLoS ONE 3:e2815; Abelson H et al. (2000) Amorphous computing. Communications of the ACM 43:74-82). Practically, researchers have begun to use enzymes that modify DNA, typically site- specific recombinases, to study and control engineered genetic systems.
  • recombinases can catalyze strand exchange between specific DNA sequences and enable precise manipulation of DNA in vitro and in vivo (Grindley NDF, Whiteson KL, Rice PA (2006) Mechanisms of site-specific recombination. Annu Rev Biochem 75:567-605 ). Depending on the relative location or orientation of recombination sites three distinct recombination outcomes, integration, excision or inversion, can be realized.
  • Single-write architectures are limiting if many of the uses for genetic data storage are considered in detail.
  • studies of replicative aging in yeast or human fibroblasts typically track at least 25 or 45 cell division events prior to the onset of senescence, respectively (Steinkraus KA, Kaeberlein M, Kennedy BK (2008) Replicative aging in yeast: the means to the end. Annu Rev Cell Dev Biol 24:29-54).
  • Lineage mapping during worm development frequently tracks at least 10 differentiation events (Sulston JE, Schierenberg E, White JG, Thomson JN (1983), The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol 100:64-119), while research with mouse and human systems considers up to several hundred cell divisions (Frumkin D, Wasserstrom A, Kaplan S, Feige U, Shapiro E (2005)
  • aspects of the present invention relate to methods and systems for rewritable digital data storage in live cells.
  • binary digits can be stored in chromosomes, which enables combinatorial data storage.
  • a DNA element can be flipped within the chromosome of a live cell, and then be flipped back to its original state. These two steps can be repeated an infinite number of times, thereby creating a binary digit (bit) data register.
  • bit binary digit
  • Such rewritability of passive (requiring no gene expression or active cellular process) data storage system is for the first time achieved by the present invention via a DNA encoded state register.
  • a combinatorial data storage having up to 2 A N bits (if N registers) can be built, which is a drastic increase from previous storage size of N bits.
  • it is also possible to support multiplexing of data storage e.g., re -use of the same recombinase enzymes to store >1 bit, such as 1 byte (8 bits)). This is useful for storing information about events inside cells (e.g., cell division or
  • environmental information that impacts cells e.g., cytokine levels, environmental pollutants, etc.
  • methods of the present invention enable the development a recombinase addressable data (RAD) modules that can be used to write and store binary digits within the chromosome of live cells.
  • RAD recombinase addressable data
  • So produced RAD systems are capable of passive and stable information storage over at least 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data structures. Additionally, by varying the synthesis and degradation rates of recombinase functions programmed
  • serine recombinase can be used.
  • the serine recombinase functions used here do not require cell-specific co-factors and can be used to extend computing and control methods to the study and engineering of many biological systems.
  • the invention provides an in vivo data storage system and methods for storing data using such a system.
  • a system includes a recombinase addressable data module comprising an invertible DNA data register.
  • the DNA data register comprises a DNA register sequence flanked by oppositional attachment sites.
  • the directionality of the DNA register sequence is invertible to a set state 1 , and optionally reversibly invertible from said set state 1 to a reset state 0.
  • the system further comprises a set generator comprising a first gene encoding an integrase; a reset generator comprising a second gene encoding an excisionase.
  • one pair of set generator and reset generator can represent one binary digit (1 bit); multiple unique pairs (N) of set generators and reset generators can be present in one system which is capable of storing up to 2 A N bits of data.
  • the reset generator further comprises a third gene encoding an integrase.
  • the attachment sites of the invertible DNA data register are recognized and recombined by the integrase of the set generator when the DNA register sequence is in reset state 0.
  • the attachment sites of the invertible DNA data register are recognized and recombined by the integrase-excisionase complex of the reset generator when the DNA register sequence is in set state 1.
  • the integrase and excisionase can be derived from bacteriophage Bxbl, TP901-1, Phirvl and/or PhiC31, or other sources.
  • aspects of the invention may include a reset generator in which the second and third genes together encode an excisionase-integrase complex or a fusion protein comprising the excisionase and the integrase.
  • at least one of the first, second, or third gene is inducible.
  • the inducible gene may be directly or indirectly induced by an inducer.
  • an inducer may directly or inactivate transcription, or indirectly or directly inactivate a repressor of transcription.
  • an inducer may require one or more co-factors.
  • aspects include an inducer that is a small molecule, a chemical, a protein, an enzyme, a nucleic acid, or a metal ion.
  • the inducer is an endogenous inducer; and in other embodiments, the inducer may be exogenous.
  • at least one of the first, second or third genes is inducible by a transcription factor; or by two or more inducers functioning together or in the alternate. Aspects also include at least one of the first, second or third genes being autoinducible.
  • Another embodiment provides a vector comprising an in vivo data storage system PCT Application
  • a recombinant cell comprising an in vivo data storage system.
  • the in vivo data storage system may be present, in whole or in part, in a chromosome of the recombinant cell.
  • the DNA data register is present in a chromosome of the recombinant cell.
  • at least one of the first, second or third genes is present in a chromosome of the recombinant cell.
  • aspects of the invention also include a method for storing data in a cell.
  • the method comprises providing a cell comprising an in vivo data storage system having a recombinase addressable data module including (i) a set generator comprising a first gene encoding an integrase; and (ii) an invertible DNA data register comprising a DNA register sequence flanked by oppositional attachment sites, wherein the directionality of the DNA register sequence is invertible to a set state 1 , and optionally reversibly invertible from said set state 1 to a reset state 0.
  • a recombinase addressable data module including (i) a set generator comprising a first gene encoding an integrase; and (ii) an invertible DNA data register comprising a DNA register sequence flanked by oppositional attachment sites, wherein the directionality of the DNA register sequence is invertible to a set state 1 , and optionally reversibly invertible from said set state 1 to a reset
  • methods according to the invention comprise inducing the first gene or allowing the induction of the first gene to express the integrase so as to allow the DNA register sequence to invert, thereby generating a set state of directionality for the DNA register sequence, the set state represented by binary digit 1, thereby storing data represented by the binary digit 1 in the cell.
  • the recombinase addressable data module further comprises a reset generator comprising a second gene encoding an excisionase and, optionally, a third gene encoding an integrase
  • the method further comprises: inducing the second gene or allowing the induction of the second gene to express the excisionase so as to allow the DNA register sequence to invert back, thereby generating a reset state of directionality for the DNA register sequence, the reset state represented by binary digit 0, thereby storing data represented by the binary digit 0 in the cell.
  • methods of the invention comprise optionally repeating step the inversion step, thereby storing data represented by the binary digit 1 or 0 in the cell.
  • aspects of the invention may relate to controlling expression of the excisionase to provide a stoichiometric amount of the excisionase in relation to one or both of an amount of an integrase and a copy number of the DNA register sequence, so as to favor generation of the reset state 0 of the DNA register sequence.
  • Methods according to the invention may further comprise tunably controlling the reversible inversion of the DNA register sequence between a state 0 and a set state 1.
  • Some embodiments comprise the method comprising one or more of: minimizing spontaneous inversion to set state 1 ; minimizing during the inversion step interference by PCT Application
  • methods of the invention comprise minimizing spontaneous inversion to set state 1 by controlling basal expression of the integrase below a threshold level for spontaneous inversion.
  • methods of the invention include minimizing during the inversion step interference by excisionase to favor generation of the set state 1 by comprises increasing degradation of the excisionase.
  • methods of the invention comprising minimizing during the reverse inversion step stoichiometry mismatch to favor generation of state 0 by one or more of: increasing expression of the excisionase, decreasing expression of the integrase, increasing degradation of the integrase, and reducing a copy number of the DNA register sequence.
  • aspects of the invention also relate to an in vivo data storage system which is a nonvolatile data storage system, and methods of storing data using a nonvolatile in vivo data storage system.
  • the DNA data register stores data as a nonvolatile memory.
  • the stored data is retained until the DNA data register rewritten or recoded with different data, erased (returned to original state), or otherwise rendered unreadable.
  • the stored data is retained in the absence of expression of the first gene.
  • the stored data is retained in the absence of expression of the first gene, the second gene, or both the first and second genes.
  • Other embodiments may provide in vivo data storage system which is a volatile data storage system and methods of recording data using volatile in vivo data storage systems.
  • Figures 1A-1D Architecture, mechanisms, and operation of a recombinase addressable data (RAD) module.
  • RAD recombinase addressable data
  • Figures 2A-2G Independent set and reset operations plus long-term data storage and switching in vivo.
  • Figures 3A-3C Functional composition, expected operable ranges, and RESET failure modes for a RAD module.
  • Figures 4A-4C Optimized genetic elements and reliable multi-cycle operation of a DNA- inversion RAD module.
  • Figures 5A-5I Maps of certain constructs used.
  • Fig. 5 A The PBAD-Int set flipper where Bxbl integrase was cloned downstream of the PBAD/AraC promoter (BBa_I0500) on pSB3Kl plasmid bearing a pl5A origin of replication (15-20 copies).
  • Fig. 5B The PBAD- Xis/Int reset flipper circuit.
  • Fig. 5C The screening vector and Fig. 5D, The RAD module depicted in Fig. 4A.
  • Figures 6A-6B Alternate architecture for a reset circuit.
  • Fig. 6A Schematic diagram of the decoupled reset circuit where integrase is expressed from a low-copy plasmid while excisionase is expressed from a medium-copy plasmid.
  • Fig. 6B Cells bearing the chromosomal LR DNA register were transformed with both plasmids encoding integrase and excisionase, pulsed with arabinose and analyzed by flow cytometry. Cells relaxed to the BP state after induction with approximately 85% efficiency.
  • Figures 7A-7C Influence of register copy number on recombination efficiency and consequences for integrase-excisionase mechanism.
  • Fig. 7A Influence of copy number of the DNA register on the efficiency of integrase-excisionase mediated recombination.
  • the bidirectional reset generator form Fig. 2C was transformed in cells containing the DNA data register in the LR state on a pSClOl plasmid (upper panel) or integrated in the chromosome (lower panel), and cells were pulsed with arabinose. When the register was on the chromosome, cells were driven toward the BP state more efficiently during induction, and the recombination efficiency of the Int/Xis reaction for LR to BP reaction was higher after inducer removal.
  • Figure 8 Screening of different RBS designed using the RBS calculator. Constructs were tested using a DNA register that expresses gemini when flipped. Cells were co-transformed with the target and the different RBS variants constructs. Cells were grown with or without
  • Figures 9A-9D Effect of the down-regulation of excisionase on set and reset functions.
  • Fig. 9A schematic representation of the RAD module used for this particular experiment.
  • the DNA data register has only one output, GFP, in the LR state.
  • the PLtet-O-1 promoter controls the set integrase while the PBAD promoter controls a polycistron expressing integrase and excisionase.
  • Both set and reset circuits are cloned on pSB3Kl plasmid (pl5A origin, 15-20 PCT Application
  • Fig. 9B control experiments.
  • Left panel BP and LR Target constructs on pSB4A5 low- copy plasmid (5-10 copies), showing the 2 states of the system (low or high GFP for BP and LR, respectively).
  • Right panel a RAD module with no copy of the excisionase was transformed in cells containing the DNA data register in the BP state on pSB4A5 and the set generator was induced with Ate. Cells flipped to the LR state as monitored by GFP expression.
  • Fig. 9C reduction of interference by down regulation of excisionase basal levels.
  • a reset generator in which the excisionase is down-regulated with an AAK ssrA tag acts as a set generator and can flip from BP to LR due to stoichiometry mismatch and higher integrase levels.
  • Figures 1 OA- IOC Example of an efficient reset generator setting-back after the end of a pulse.
  • Fig. 10A Kinetic model based simulation of the reset efficiencies during and after the pulse for different expression scaling.
  • a gray dot marks an integrase- excisionase expression scales that keeps the latch in an intermediate state both during and after a RESET pulse; a black dot (in white circle) marks an expression scale that causes the latch to revert back to LR state after a reset pulse.
  • Fig. 10B Time-course simulation showing resetting failures during and after a RESET pulse. Gray and black lines are simulated using integrase and excisionase expression scaling parameters as marked with gray and black dots in Fig. 10A.
  • Fig. IOC Schematic representation of the particular construct used in Fig.
  • excisionase has a strong RBS and AAK degradation tag while integrase has a very weak RBS (BBa_B0033) followed by a GTG start codon. Therefore, even if the stoichiometry between the two proteins is correct during the pulse, the degradation rate of excisionase is higher than for integrase, resulting in entry into the set regime after the pulse.
  • Figures 1 lA-1 IB Example of a set circuit resetting back after the end of a pulse.
  • Fig. 11 A Kinetic model based simulation of the reset efficiencies during and after the pulse for different expression scaling.
  • a gray dot in white circle) marks an integrase-excisionase PCT Application
  • Figure 12 Detailed parameter sensitivity analysis of DNA inversion RAD module operable range, (i), Kinetic model based simulation of the set, reset and SR-latch efficiencies after the pulse for different expression scaling and kinetic parameters.
  • the integrase lower bound of the operable range scales with integrase-flipper dissociation constant (ii), the excisionase upper bound of the set operable range and the excisionase lower bound of the reset operable range scale with integrase-excisionase dissociation constant (iii), the excisionase lower bound of the reset operable range and the integrase lower bound of the set and the reset operable range scale with the fold change between induced and basal expression (iv) increasing the amount of DNA register changes the excisionase upper bound of set operable range and the excisionase lower bound of reset operable range.
  • Figures 13A-13F Detailed data for the RAD module operation cycles.
  • Fig. 13C Quantification of switching plus storage efficiency of the RAD module for long input cycles.
  • Fig. 13D Quantification of switching plus storage efficiency of the RAD module for short input cycles.
  • Figs. 13C and 13D error bars are PCT Application
  • Fig. 13E long term storage of the BP state in the context of the RAD module.
  • Cells in the LR state were RESET with arabinose, switched and stored the BP state. After 40 generation, cells were SET back with Ate (blue/open circle line) or grown without inducer up to 100 generations of storage (red/filled square line).
  • Fig. 13F long term storage of the LR state in the context of the RAD module. After one cycle of RESET with arabinose and SET with Ate, cells were grown for 40 generations, RESET with arabinose or grown without inducer up to 100 generations. In both Figs. 13E and 13F, switching plus storage efficiency was comparable with the initial efficiencies.
  • Figures 14A-14C Comparison of operable ranges of S/R latches using alternative mechanisms.
  • Fig. 14A Schematic diagram of a hypothetic S/R latch based on a DNA inversion RAD module whose DNA register can be inverted and reverted by two different recombinase, Reel and Rec2 respectively.
  • Fig. 14B Schematic diagram of a mutual inhibition S/R- latch. A pair of mutually repressed genes functions as a bistable switch; an additional copy of each gene, driven by SET or RESET inputs, is necessary to couple arbitrary transcriptional signals to the state of the bistable switch.
  • Fig. 14C Kinetic model based simulation of the set, reset and SR- latch. Note that the operable range for set and reset circuits are symmetric and there is no efficiency loss due to stoichiometry mis-match as in the case of integrase-excisionase based DNA inversion latch.
  • Figures 15A-15B Tuning the reset-specific integrase degradation rate allows for different bias in the outcome of integrase-excisionase mediated recombination.
  • Fig. 15 A Schematic representation of the system used in this experiment. Only the integrase ssrA degradation tag changes. These constructs are alternate resets elements obtained during the screen for a functional reset using a destabilized excisionase.
  • Fig. 15B Three resets elements using different ssrA sequences for the integrase display different proportions of cells in the BP state after pulse (pink: gate used to measure the percentage of cells in BP along with the actual value).
  • Figures 16A-16Q Maps of various genetic elements used herein. Fig. 16A,
  • Fig. 16F PhiC31PbadXisPtetIntj64100.
  • Fig. 16G Phirvl constIntj64100.
  • Fig. 16H Phirvl PbadXisPtetlnt integrationVector.
  • Fig. 161 PhiC31PbadXisPtetInt_integrationVector.
  • Fig. 16F PhiC31PbadXisPtetIntj64100.
  • Fig. 16G Phirvl constIntj64100.
  • Fig. 16H Phirvl PbadXisPtetlnt integrationVector.
  • Fig. 161 Phirvl PbadXisPtetlnt integrationVector.
  • TP901-1, Phirvl and PhiC31 Cells bearing chromosomal BP data register built from attB/attP recombination sites of bacteriophage Bxbl, TP901-1, Phirvl or PhiC31 weree transformed with a medium copy plasmid expressing their cognate integrases. Expression of the integrase drove state switching of the data register from BP state to LR state (arrows indicate switching from gray/before switching to black/after switching).
  • Figure 18 Independent reset operations using serine integrases and excisionases from bacteriophage Bxbl, TP901-1, Phirvl and PhiC31.
  • Cells bearing chromosomal LR data register built from attL/attR recombination sites of bacteriophage Bxbl, TP901-1, Phirvl or PhiC31 were transformed with a medium copy plasmid expressing their cognate integrases and excisionases.
  • Coexpressing integrase and excisionase drove state switching of the data register from LR state toward BP state (arrows indicate switching from gray/before switching to black/after switching).
  • FIG. 19 Specificity of integrase- DNA data register from bacteriophage Bxbl, TP901- 1, Phirvl and PhiC31.
  • Cells bearing chromosomal BP data register from bacteriophage Bxbl, TP901-1, Phirvl or PhiC31 were transformed with a medium copy plasmid expressing an integrase from bacteriophage Bxbl, TP901-1, Phirvl or PhiC31 (represented by black dots; negative control is represented by gray dots where there is no integrase expression). Only when the integrase and the data register are from the same bacteriophage can the integrase efficiently switch the data register from BP state to LR state (i.e., black dots that do not overlap with gray dots).
  • Figure 20 Resetting by RAD modules with both integrase and excisionase expressed from chromosome.
  • the PBAD-Xis / Ptet-Int flipper circuit was integrated to Escherichia coli chromosome at phage HK022 integration site; its cognate LR data register was integrated at phage phi80 site (Bxbl and PhiC31) or at phage p21 site (Phirvl). Dual induction with 0.1% arabinose and 200 ng/ml drove state switching from LR to BP state.
  • RAD rewriteable recombinase addressable data
  • phage integrases are unique in that the directionality of the recombination reaction can be influenced by an excisionase co- factor (Groth AC, Calos MP (2004) Phage integrases: biology and applications. Journal of Molecular Biology 335:667-678).
  • Groth AC Calos MP (2004) Phage integrases: biology and applications. Journal of Molecular Biology 335:667-678.
  • a phage integrase alone typically catalyzes site-specific recombination between an attP site on the infecting phage chromosome and an attB site encoded within the host chromosome.
  • the resulting integration reaction inserts the phage genome within the host chromosome bracketed by newly formed attL and attR (LR) sites.
  • Phages integrases are thought to represent two evolutionary and mechanistically distinct recombinase families (Groth AC, Calos MP (2004) Phage integrases: biology and applications. Journal of Molecular Biology 335:667-678).
  • Tyrosine integrases such as the bacteriophage lambda integrase, often have relatively long attachment sites (-200 bp), use a Holliday junction mechanism during strand exchange, and require host specific co-factors.
  • serine integrases use a double-strand break mechanism during recombination and can have shorter attachment sites (-50 bp).
  • some serine integrases do not require host cofactors, a feature that has led to their successful reuse across a range of organisms (Keravala A et al.
  • bacteriophage serine integrase was used in the examples provided herein.
  • suitable bacteriophage serine integrase include but not limited to, Hin, Gin, Cin, cpC31, cpRvl, R4, TP901, A118, U153, Bxbl and cpFCl .
  • Bacteriophage Bxbl now provides the best characterized serine integrase excisionase system (Kim AI et al. (2003) Mycobacteriophage Bxbl integrates into the Mycobacterium smegmatis groELl gene. Molecular Microbiology 50:463-473; Ghosh P, Kim AI, Hatfull GF (2003) The orientation of mycobacteriophage Bxbl integration is solely dependent on the central dinucleotide of attP and attB. Molecular Cell 12: 1101-1111; Ghosh P, Wasil LR, Hatfull GF
  • Bxbl gp35 is a serine integrase that catalyzes integration of the Bxbl genome into the GroELl gene of M. smegmatis (Kim AI et al. (2003) Mycobacteriophage Bxbl integrates into the Mycobacterium smegmatis groELl gene. Molecular Microbiology 50:463-473).
  • Bxbl gp47 is an excisionase that mediates excision in vivo and has been shown to control recombination directionality in vitro with high efficiency (Ghosh P, Wasil LR, Hatfull GF (2006) Control of phage Bxbl excision by a novel recombination directionality factor. PLoS Biol 4:el86).
  • Minimal attB, attP, attL, and attR sites have been defined for the Bxbl system (Kim AI et al. (2003) Mycobacteriophage Bxbl integrates into the Mycobacterium smegmatis groELl gene.
  • the Bxbl excisionase does not bind DNA independently and, from in vitro studies, is thought to control integrase directionality in a stoichiometric manner (Ghosh P, Wasil LR, Hatfull GF (2006) Control of phage Bxbl excision by a novel recombination directionality factor. PLoS Biol 4:el86).
  • Mycobacterium tuberculosis H37Rv another serine integrase, excisionase and att sites were identified (Bibb LA, Hatfull GF (2002) Integration and excision of the Mycobacterium tuberculosis prophage-like element, phiRvl . Mol Microbiol 45(6): 1515-26).
  • Bibb LA Hatfull GF (2002) Integration and excision of the Mycobacterium tuberculosis prophage-like element, phiRvl . Mol Microbiol 45(6): 1515-26).
  • PhiC31 excisionase can bind to its cognate integrase in the absence of recombination sites.
  • the set element for Bxbl (PBAD-driven-integrase generator, Figs. 2B, 2D, 2E) was cloned in pSB3Kl plasmid (pl5A origin; 15-20 copies).
  • the reset element for Bxbl (PBAD- driven-excisionase+integrase generator, Fig. 2C, 2D, 2E) was cloned on J64100 plasmid.
  • the full RAD module (PLtet-Ol driven integrase generator and PBAD-driven-excisionase+integrase generator, Fig. 4, Fig. 14) was cloned in J64100.
  • PBAD-driven excisionase / Ptet-driven integrase generator (Fig. 17 Bxbl, TP901-1 and PhiC31, Fig. 18) was cloned in J64100 plasmid.
  • Constitutive promoter-driven integrase generator (Fig. 17 Phirvl, Fig. 19) was cloned in J64100 plasmid.
  • L- arabinose (Calbiochem) was used at a final 0.5% w/v concentration; anhydrotetracycline (Sigma) was used at a final concentration of 20 ng/ml.
  • Figs. 17-20 cells were grown in Hi-Def Azure medium (Teknova) supplemented with 0.66 % v/v glycerol.
  • L-arabinose was used at a final 0.2%> (Fig. 17 and 18) or 0.1% (Fig. 20) w/v concentration; and hydrotetracyclin was at a final concentration 200 ng/ml.
  • a saturated culture was diluted 1 :2000 in media with inducer.
  • Cells were centrifuged and washed before each dilution step.
  • overnight grown cultures were diluted 1 : 100 in media with inducer, grown for 4 hours, at which point cells were washed, diluted 1 :2000, and grown for an additional 16H. For Figs. 17-20 induction was done overnight.
  • RAD Recombinase Addressable Data
  • the RAD module consists of an inducible "set” generator producing integrase, an inducible “reset” generator producing integrase and excisionase, and a DNA data register (Fig. 1 A).
  • An alternative RAD module architecture used in Fig. 17 (Bxbl, TP901-1 and PhiC31) and Fig. 18 has integrase and excisionase under two different inducible promoters (Ptet and PBAD, respectively) so that integrase and
  • the DNA inversion RAD module is driven by two generic transcription input signals, set and reset.
  • a set signal drives expression of integrase that inverts a DNA element serving as a genetic data register. Flipping the register converts flanking attB and attP sites to attL and attR sites, respectively.
  • a reset signal drives expression of integrase and excisionase and restores both register orientation and the original flanking attB and attP sites.
  • the register itself encodes a constitutive promoter which initiates strand-specific transcription. Following successful set or reset operations, mutually exclusive transcription outputs "1" or "0" are activated, respectively. For the RAD module developed here a "1" or "0" register state produces red or green fluorescent protein, respectively.
  • integrase alone should set a DNA register sequence flanked by oppositional attB and attP sites thereby producing an inverted sequence flanked by attL and attR sites (State "1").
  • a second independent transcriptional input drives the simultaneous production of integrase and excisionase and should reset the register sequence to its original orientation and flanking sequences (State "0").
  • Fig. IB shows the elementary chemical reactions, molecular species, and kinetic parameters used to model the RAD module.
  • Molecular concentrations are normalized to the integrase dimer dissociation constant (K;).
  • Kinetic rates are normalized to the integrase-mediated recombination rate (k c _1 ).
  • the model reflects available PCT Application
  • Fig. 1C is a simulated phase diagram detailing pseudo equilibrium operating regimes for a RAD module experiencing sustained integrase and excisionase expression levels for 200/k c .
  • the red (left corner curves), green (right corner curves), and gray (bottom curves) lines represent, with decreasing intensity, 95, 75, and 55% switching (or hold) efficiencies.
  • Three distinct latch operating regions were found as a function of integrase and excisionase expression levels, corresponding to expected "set,” “reset,” or “hold” operations (Fig. 1C).
  • One complete latch cycle requires the dynamic adjustment of integrase and excisionase expression through a "set, hold, reset, hold" pattern. These operations are realized in practice by cycling the transcription signals that define latch set and reset inputs and by tuning the specific genetic elements that provide fine control over integrase and excisionase synthesis and degradation.
  • a data storage register was first implemented via a DNA fragment encoding fluorescent reporter proteins and Bxbl recombinase recognition sites flanking a constitutive promoter on the chromosome of E. coli DH5aZl (Lutz R, Bujard H (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/Il-I2 regulatory elements. Nucleic Acids Research 25: 1203-1210) (Fig. 1A). The state of the register could be assayed reliably was confirmed via microscopy and cytometry (Fig. 2A). In Fig. 2A, microscopy and flow cytometry data show two distinguishable states for an invertible data register integrated in the E. coli chromosome driving red (RFP) or green (GFP) fluorescent proteins, and also a control sample in which cells express neither reporter. It was next established that the register PCT Application
  • the set-encoding vectors were transformed into cells containing the chromosomal BP register and isolated cells that only switched when induced; many variants switch spontaneously in the absence of an input signal or do not switch when induced (Fig. 8, Table 2).
  • Set functions were isolated that switch with greater than 95% efficiency at the single cell level and that hold state following inducer removal (Fig. 2B).
  • Fig. 2B data register inverses via expression of integrase. Growing cells (doubling time -90 min) start in state "0" expressing GFP and, following a 16 hour set input pulse, switch to and hold state "1" expressing RFP.
  • Fig. 2C shows bidirectionality of the integrase-excisionase reaction.
  • Cells were transformed with plasmids containing the LR DNA data register and a bi-directional reset element on a plasmid and pulsed with arabinose. During a pulse, cells entered an intermediate state where both GFP and RFP are expressed. After inducer removal, cells split into two major populations corresponding to BP and LR states. Split BP and LR populations were sorted by FACS and pulsed these sorted cells with arabinose again. The same behavior was observed regardless of the initial register state.
  • 2D shows the stochastic simulation of bidirectional DNA inversion for a single copy DNA register (top row) before, during (blue shaded area) and after a reset pulse.
  • BP to LR and LR to BP recombination propensities are assumed to be equal.
  • Two independent time-course stochastic simulations (middle and bottom rows) of expected GFP and RFP expression levels given the depicted (top row) BP and LR states.
  • Fluorescent reporter degradation propensities modeled as ten-fold slower than recombination propensities. From this framing, engineer reset controllers were then engineered that produce a range of weighted outcomes in the final register state by tuning the reset-specific integrase degradation rate (25, 50, 75% BP:LR distributions; Fig. 15).
  • the fraction of integrase available for excisionase binding may be decreased and therefore the effective excisionase-to-integrase ratio may increase.
  • the fraction of integrase available for excisionase binding may be decreased and therefore the effective excisionase-to-integrase ratio may increase.
  • the DNA register copy number from 5-10 per cell (Fig. 2C) to 1 per cell (Fig. 7A, bottom) an increase in excisionase-mediated recombination directionality from -40% to -65% was observed.
  • Such observations are consistent with a model that accounts for relative DNA copy number and whether excisionase can interact with cytoplasmic integrase (Figs. 7B-7C).
  • E. coli cells were repetitively grown and diluted every day for 10 days and monitored data storage at the single cell level by measuring the continuous expression of fluorescent reporters. It was established that starting from either state the register could switch and hold state for 100+ cell doublings (Fig. 2F) or could hold state and then switch reliably following 90+ cell doublings (Fig. 2G). Fig. 2F shows stable long term data storage. Cells were serially propagated without input signals for 100 generations following data register set (orange) or reset (blue).
  • Fig. 2G shows long term functionality of data register. Cells were serially propagated without input signals for 90 generations and then exposed to set (orange) or reset (blue) input signals. The fraction of individual cells switching state was assayed by cytometry. Taken together these data demonstrate the practical stability and long-term PCT Application
  • Fig. 3A shows simulated phase diagrams detailing the expected operation of RAD module functions in response to dynamic pulses of integrase and excisionase across a range of expression levels.
  • Top row switching efficiencies (colormap) for set and reset functions when combined in a RAD module.
  • Fig. 3B shows reset failure during a reset pulse due to stoichiometric mismatch-mediated bidirectional register switching.
  • Fig. 3C shows reset failure immediately following a reset pulse due to stoichiometric mismatch-mediated setting to state "1".
  • Fig. 4A shows details of an integrated DNA inversion RAD module optimized for reliable set, reset and storage functions. Specific genetic regulatory elements controlling protein synthesis and degradation were obtained from standard biological parts collections (filled shapes with black outline such as AAK and B0031) (e.g., those from the Registry of Standard Biological Parts at MIT, as well as the International Open Facility
  • FIG. 4B shows experimental RAD module operation over multiple duty cycles. Growing cells (doubling time -90 minutes) starting in state "1" were cycled through a "reset (marked by "R”), hold (marked by "St”), set (marked by "S”), hold” input pattern with each step lasting -10
  • Fig. 4C shows multi-cycle RAD module PCT Application
  • Atty Docket No. 062602-021402/PCT operation driven by shorter SET and RESET input pulses. As in Fig. 4B but with set and reset pulses lasting for ⁇ 2 cell doublings ( ⁇ 3 hours).
  • Three additional exemplary data storage registers were implemented via a DNA fragment encoding fluorescent reporter protein and TP901-1, Phirvl and PhiC31 recombinase recognition sites flanking a constitutive promoter on the chromosome of E. coli DH5aZl .
  • these three data storage registers can be switched from a BP state to an LR state using their cognate integrase (Fig. 17) and from an LR state to a BP state using their cognate integrase-excisionase (Fig. 18) expressed from J64100 plasmids.
  • the RAD device design can have the input module (the transcriptional sources, integrase and excisionase generator) on a medium copy plasmid in order to produce enough integrase and excisionase for efficiently switching the state of the output module (recombination site and output promoter) on a genomic DNA, especially during reset.
  • functional composition of multiple RAD units may require an ability to connect the output module of one RAD unit to the input module of the other RAD unit.
  • integrase and excisionase generators were integrated to HK022 site on E.coli chromosome. Integrase was expressed under Ptet while excisionase was expressed under PBAD.
  • Table 2 summarizes certain failure modes and engineering solutions for set and reset operations. Putative failure causes as noted.
  • Expression cassette schematics highlight (orange) regulatory regions targeted for reengineering with element-specific redesign goals.
  • 6N is a full six nucleotide library within a Shine -Dalgarno ribosome binding site (RBS) core.
  • RBS-1 and RBS-2 are collections of "standard” or computationally designed RBSs.
  • AXX is a peptide library sampling 12 biochemically representative amino acids. The number of independent clonal constructs tested in each case plus corresponding figures are provided.
  • conditional control over recombination directionality to implement a repeatedly rewritable DNA data storage element likely only partially aligns with the natural contexts in which integrase and excisionase performance have been selected.
  • integrase alone naturally mediates integration of a phage genome into a host chromosome under circumstances in which the phage will not destructively lyse the host cell.
  • Such integration reactions are likely under positive selection to be fast and efficient, given that failure to integrate prior to host chromosome replication and cell division could result in loss of the phage from a daughter lineage. Integration reactions are also likely under negative selection to be irreversible, since integration followed by immediate excision could result in an abortive infection.
  • the DNA inversion RAD module developed here should be translatable to applications requiring stable long-term data storage (for example, replicative aging) or under challenging conditions (for example, clinical or environmental contexts requiring in situ diagnosis or ex post facto reporting via PCR or DNA sequencing). Given the natural phage recombination functions from which the latch is implemented (Ringrose L et al. (1998) Comparative kinetic analysis of FLP and ere recombinases: mathematical models for DNA binding and recombination.
  • RDFs recombination directionality factors
  • Plasmids were constructed using standard BioBrickTM (Knight T (2003) Idempotent Vector Design for Standard Assembly of Biobricks) or Gibson assembly (Gibson DG et al. (2009)
  • RG (1981) Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. Proc Natl Acad Sci USA 78:752-756) (BBa_I0500), Superfolder GFP (Pedelacq J-D, Cabantous S, Tran T, Terwilliger TC, Waldo GS (2005) Engineering and characterization of a superfolder green fluorescent protein.
  • Bxbl integrase was cloned on pSB4A5 plasmid (pSC 101 origin, 5-10 copies (Shetty RP, Endy D, Knight TF (2008) Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2:5) while the excisionase was cloned on J64100 plasmid (regulated ColEl; 50-70 copies). Sequences are available via GENBANK accession numbers JQ929581 to JQ929585 and via the MIT Registry of Standard Biological Parts.
  • the DNA data register in BP and LR states consist of a constitutive promoter
  • BBa_J23119 flanked by BP or LR recombination sites positioned in opposite orientation, resulting in DNA inversion when recombined (Figs 1 & 2 and Figs 5E and 5F).
  • a Rrnp Tl terminator (BBa_J61048) was added in reverse orientation upstream of the promoter to prevent transcriptional read-through in the opposite orientation, so that in each state, only one fluorescent protein is visibly expressed.
  • superfolder GFP and mKate2 were cloned (Shcherbo, D. et al. Far-red fluorescent tags for protein imaging in living tissues. Biochem. J.
  • Bxbl integrase was cloned downstream of the PBAD/AraC promoter (BBa_I0500) on pSB3Kl plasmid bearing a pl5A origin of replication (15-20 copies).
  • This version of the integrase has a 6-His-tag which was found to stabilize the protein. Therefore, a weak RBS (BBa_B0031) and a LAA ssrA tag was added to reduce the basal expression of the enzyme.
  • PBAD controls expression of a polycistron encoding excisionase with a strong RBS designed using the RBS Calculator (Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946-950) to have a target Translation Initiation Rate (TIR) of 50000, followed by Bxbl integrase with a GTG start codon to decrease its TIR (Barrick, D., Villanueba, K., Childs, J. & Kalil, R. Quantitative analysis of ribosome binding sites in E. coli.
  • RBS Calculator Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946-950
  • TIR Target Translation Initiation Rate
  • PBAD-Bxbl integrase was cloned (with no 6-His tag and no Ssra tag) on a pSClOl plasmid pSB4A5 (5-10 copies), and transformed it in cells containing the DNA data register in LR state along with PCT Application
  • Plasmids were transformed in chemically competent E. Coli DH5alphaZl and plated on LB agar plates containing the appropriate antibiotics.
  • the set circuit presented in Fig. 2B was transformed in cells containing HK022 integrated BP DNA register bearing a chloramphenicol resistance cassette.
  • the polycistronic reset circuit, the decoupled reset circuit, and all S/R RAD modules were transformed in cells containing Phi80-integrated BP or LR DNA register bearing a kanamycin resistance cassette.
  • all DNA register bears a kanamycis resistance cassette.
  • Bxbl, TP901-1 and PhiC31 data register were integrated at Phi80 site; Phirvl data register was integrated at p21 site.
  • Cells containing the chromosomal DNA data register were grown with an additional 5 ⁇ g/ml of kanamycin or chloramphenicol depending on the integrated cassette.
  • a screening vector (Fig. 5C) was built containing the excisionase fused to an AAK Ssra tag under the control of the arabinose promoter.
  • a PLtetO-1 promoter followed by Hindlll and Nsil sites allows for cloning and screening of Ate controlled set circuits.
  • the excisionase gene is followed by Ascl and the BioBrickTM suffix Spel and Pstl sites, allowing for cloning of a reset integrase.
  • the library was built by amplifying the Bxbl integrase gene with forward primers containing B0031 RBS and a GTG start codon and reverse primers containing the randomized ssrA tag, and cloning the PCR library into the screening vector in between the Ascl and Spel sites. Ligations were transformed into DH5alphaZl cells containing the chromosomal LR DNA register. 384 clones, corresponding to almost 95% coverage of the library (Reetz, M.T., Kahakeaw, D. & Lohmer, R. Addressing the numbers problem in directed evolution.
  • Gemini a bifunctional enzymatic and fluorescent reporter of gene expression.
  • PLoS ONE 4, e7569 (2009) a bifunctional reporter containing the alpha-fragment and GFP, downstream of the invertible promoter. Therefore, recombinase activity can be monitored by beta-galactosidase activity.
  • X-Gal was used at a final concentration of 70 ⁇ g/mL concentration and IPTG at a 80uM final concentration.
  • the Bxbl integrase gene was PCR amplified using primers containing a RBS with a randomized Shine-Delgarno sequence and a GTG start codon (SEQ ID NO.:23:
  • ODE Ordinary differential equation
  • the model consists of three components: integrase (I), excisionase (X) and DNA register (D).
  • BP to LR recombination is catalyzed by an integrase tetramer (a complex of integrase dimer binding to attB and attP sites).
  • LR to BP recombination is catalyzed by an integrase-excisionase complex, integrase-excisionase stoichiometry in the complex for Bxbl is unknown but is assumed to be 1 : 1.
  • Fig. 1 C the dynamics of total register in an LR state, D L Rtot, can be written as: PCT Application
  • concentration of each complex can be written as:
  • Ki , Kj; and K d i x are dissociation equilibrium constants of integrase-integrase dimer, of integrase dimer-recombination site complex and of integrase-excision complex on a
  • the dual recombinase RAD module model (Fig. 14A) consists of three components: recombinase-1 (Rl), recombinase-2 (R2) and DNA register (D).
  • Rl recombinase-1
  • R2 recombinase-2
  • D DNA register
  • K c is an inversion rate constant and K
  • K d i are dissociation equilibrium constants of recombinase dimer and recombinase dimer-recombination site, respectively.
  • Binary state could be stored epigenetically using a bistable gene regulator network, for example, a system of two mutually repressing genes (Gardner TS, Cantor CR, Collins J J, (2000)
  • the network can be set and reset simply by adding external inducers (IPTG, aTc, heat shock, etc.) that can inactivate one of the two repressors, allowing the other repressor to express.
  • IPTG external inducers
  • aTc heat shock, etc.
  • the mutual inhibition S/R latch model presented here (Fig. 145) consists of repressor Rl and R2 mutually repressing each other expression and an extra copy of Rl and R2 driven by set input and reset input, respectively. Assuming that repressors bind to their cognate operator sites as tetramer (as it was assumed for recombinases), the dynamics of Rl and R2 concentrations can be written as: j3 ⁇ 4 + [R2 i 4 PCT Application
  • Total DNA register concentration is 1 (in Kiunit).
  • the state storage element allows the latch to maintain the state; this includes a DNA register of the DNA inversion RAD module or a bistable mutual inhibition circuit of a mutual inhibition S/R latch.
  • a DNA register encodes a state in a DNA sequence which is naturally maintained and replicated inside living cells; a mutual inhibition circuit encodes a state as repressor concentration which can be maintained through a feedback loop.
  • the input interface element allows external inputs (transcriptional signals for examples presented here) to perturb and change the state of the state storage element. This includes the integrase-excisionase genes (or dual recombinases) for the DNA inversion RAD module or extra copies of repressor genes driven by inputs, for a mutual inhibition S/R latch.
  • a challenge in implementing a RAD module is to properly "map" the dynamic ranges of the external input of interest, via the input interface element, to the state phase of the state storage element.
  • mapping is represented as the expression scaling parameter ⁇ .
  • ⁇ is proportional to translation rate and inverse proportional to protein degradation rate.
  • Another challenge for implementing a RAD module is to optimize two antagonizing mechanisms, the set and the reset mechanisms, within the same chassis.
  • Optimal conditions for resetting of the integrase-excisionase based S/R latch, for example, having a stable and efficiently translating excisionase would be likely to have so high excisionase basal expression that can interfere with the setting mechanism.
  • the size of the operable range with respect to the scaling parameter will be proportional to the fold changes between the basal input level and the input pulse. If the fold change is small, one needs to precisely match the basal input level to state storage regime and the PCT Application
  • Both set and reset operable ranges have a lower bound of integrase expression level corresponding to the induced integrase level that is "enough" for efficient recombination.
  • the rate of BP to LR recombination is governed by the level of [DI 4 ], which can be switched to LR state, relative to [D] and [DI 2 ] which cannot:
  • Excisionase determines directionality of state switching. Too high basal excisionase expression will break a set. On the contrary, too low induce excisionase expression will break a reset. Consider how much excisionase expression scaling will allow both efficient set and reset.
  • the net BP to LR and LR to BP recombination rates depend on the relative amount of the active recombination complex, [DI 4 ] and [DI 4 X 4 ], which, in turn, depends on excisionase level: ⁇ 5 : 1 ⁇ 2] M*
  • basal excisionase level is 0.1 ⁇ ⁇ so the upper bound of PCT Application
  • Reset operable range has an additional bound: the upper bound of integrase production which is not enough for causing spontaneous BP to LR recombination at the end of the reset pulse (Fig. 10).
  • Reset becomes inefficient if [I tot ] > [Xtot] during the reset pulse because, at the end of the pulse, excisionase will disappear first and thus left over integrase will drive BP state register back to LR state again.
  • operable ranges of dual recombinase RAD module based S/R latch or mutual inhibition S/R latch with respect to input element expression scaling are constrained by (Fig. 14): 1) the expression scaling lower bound that is large enough to allow state change during a pulse, and 2) the expression scaling upper bound that small enough to not allow spontaneous state switching in the absence of an input pulse.
  • Operable range size i.e., the distance between the lower and the upper expression scaling bounds is approximately the fold changes between the induced and the basal input levels.
  • Rectangular operable range shape results from the fact that the set and the reset mechanism for the dual recombinase RAD module (or for the mutual inhibition S/R latch) do not directly interacting with each other and that there is no loss of operable regime due to stoichiometry mismatch. Note that for mutual inhibition S/R latch, there is a sharp transition between efficient and inefficient set or reset operable range due to bistability of the system.
  • Simulated RAD operable range can recapitulate experimentally observed dependence between the copy number of DNA register, the copy number of integrase-excisionase genes and S/R latch efficiency (Fig. ⁇ and IB). Specifically, increasing the copy number of DNA register relative to the copy number of integrase excisionase genes decreases resetting efficiency.
  • Parameter setting used in the simulation shown here is the same as that of the default parameter setting except for that excisionase production rate during reset is reduced to only half of integrase production rate.
  • the amount of integrase-DNA register complexes approaches the total amount of integrase. If total integrase outnumbers total excisionase, there are too many integrase-DNA register complexes for excisionase to bind to and thus BP to LR recombination cannot be suppressed completely.
  • the amount of integrase-DNA register is limited by the PCT Application
  • Fig. 3B the failure mode of a RAD module is presented in which each cell in the population expressing both GFP and RFP during an input pulse and then splitting into two populations of cells expressing either GFP or RFP after the pulse.
  • the model consists of a single copy DNA register which can be in either state 0, expressing GFP, or state 1, expressing RFP.
  • the scenario was simulated in which the net propensity for inverting from state 0 to state 1 and from state 1 to state 0 are equal. This scenario corresponds to the region between the set and the reset regime in Fig. ID. It was also assumed that the degradation propensity of the both reporters is ten times slower than the inversion propensity (it was expected that in the

Abstract

La présente invention concerne un module de données adressables par recombinase (RAD) réinscriptibles qui stocke de façon fiable des informations numériques dans un chromosome. Les modules RAD décrits utilisent les fonctions sérine intégrase et excisionase pour inverser et restaurer des séquences d'ADN spécifiques. L'élément de mémoire RAD est capable de stocker des informations passives en l'absence d'expression de gène hétérologue sur plusieurs générations, et peut être échangé de façon répétée sans dégradation des performances pour prendre en charge le stockage de données combinatoires.
PCT/US2013/040089 2012-05-08 2013-05-08 Procédés et compositions pour stockage de données numériques réinscriptibles dans des cellules vivantes WO2013169867A1 (fr)

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US10892034B2 (en) 2016-07-01 2021-01-12 Microsoft Technology Licensing, Llc Use of homology direct repair to record timing of a molecular event
CN113462710A (zh) * 2021-06-30 2021-10-01 清华大学 一种可随机重写的dna信息存储方法
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Cited By (7)

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Publication number Priority date Publication date Assignee Title
WO2014093852A1 (fr) * 2012-12-13 2014-06-19 Massachusetts Institute Of Technology Logique et systèmes de mémoire basés sur la recombinase
US9691017B2 (en) 2012-12-13 2017-06-27 Massachusetts Institute Of Technology Recombinase-based logic and memory systems
US10669558B2 (en) 2016-07-01 2020-06-02 Microsoft Technology Licensing, Llc Storage through iterative DNA editing
US10892034B2 (en) 2016-07-01 2021-01-12 Microsoft Technology Licensing, Llc Use of homology direct repair to record timing of a molecular event
US11359234B2 (en) 2016-07-01 2022-06-14 Microsoft Technology Licensing, Llc Barcoding sequences for identification of gene expression
CN113462710A (zh) * 2021-06-30 2021-10-01 清华大学 一种可随机重写的dna信息存储方法
CN113462710B (zh) * 2021-06-30 2023-07-11 清华大学 一种可随机重写的dna信息存储方法

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