Research Article

Genomically encoded analog memory with precise in vivo DNA writing in living cell populations

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Science  14 Nov 2014:
Vol. 346, Issue 6211, 1256272
DOI: 10.1126/science.1256272

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  1. Fig. 1 SCRIBE system for recording inputs in the distributed genomic DNA of bacterial populations.

    (A) Synthetic ssDNA (red lines) generation inside of living cells by retrons. (B) Visualization of retron-mediated ssDNAs produced in living bacteria. The amount of ssDNA in each sample (shown in brackets) was calculated by densitometry. (C) A kanR reversion assay was used to measure the efficiency of DNA writing within living cells, where the msd(kanR)ON cassette and the bet gene were inducible by IPTG and aTc, respectively. (D) Demonstration of analog memory achieved via SCRIBE to record the magnitude of an input into genomic DNA. The green line is a linear regression fit. The red dashed brackets marked with asterisks connect the closest data points that are statistically significant with respect to each other (P < 0.05 based on one-tailed Welch’s t test). Error bars indicate SEM for three independent biological replicates.

  2. Fig. 2 SCRIBE can write multiple different DNA mutations into a common target loci (galK).

    (A) Schematic of the procedure (see text for details). (B) galKON cells harboring the circuits shown in (A) were induced with either IPTG (1 mM) or aTc (100 ng/ml) for 24 hours, and the galKOFF frequencies in the population were determined by plating the cells on appropriate selective conditions. (C) galKOFF cells [obtained from the experiment described in (B)] were induced with IPTG (1 mM) or aTc (100 ng/ml) for 24 hours, and the galKON frequencies in the population were determined by plating the cells on appropriate selective conditions. Error bars indicate SEM for three independent biological replicates.

  3. Fig. 3 Writing multiple mutations into independent target loci within population.

    (A) Constructs used to target genomic kanROFF and galKON loci with IPTG-inducible and aTc-inducible SCRIBE cassettes, respectively. (B) Induction of kanROFF galKON cells with IPTG or aTc generates cells with the kanRON galKON or kanROFF galKOFF genotypes, respectively. Induction of kanROFF galKON cells with both IPTG and aTc generates cells with the kanRON galKOFF genotype. (C) kanROFF galKON reporter cells containing the circuits in (A) were induced with different combinations of IPTG (1 mM) and aTc (100 ng/ml) for 24 hours at 30°C, and the fraction of cells with the various genotypes were determined by plating the cells on appropriate selective media. Error bars indicate SEM for three independent biological replicates.

  4. Fig. 4 Optogenetic genome editing and analog memory for long-term recording of input signal exposure times in the genomic DNA of living cell populations.

    (A) We coupled expression of SCRIBE(kanR)ON to an optogenetic system (PDawn). The yf1/fixJ synthetic operon was expressed from a constitutive promoter: In dark conditions, YF1 interacts with and phosphorylates FixJ. Phosphorylated FixJ activates the PfixK2 promoter, which drives λ repressor (cI) expression, which subsequently represses the SCRIBE(kanR)ON cassette. Light inhibits the interaction between YF1 and FixJ, leading to the generation of ssDNA(kanR)ON and Beta expression and, thus, the conversion of kanROFF to kanRON. Cells harboring this circuit were grown overnight at 37°C in the dark, diluted 1:1000, and then incubated for 24 hours at 30°C in the dark (no shading) or in the presence of light (yellow shading). Subsequently, cells were diluted by 1:1000 and grown for another 24 hours at 30°C in the dark or in the presence of light. The dilution-regrowth cycle was performed for four consecutive days. The kanR allele frequencies in the populations were determined by sampling the cultures after each 24-hour period. (B) SCRIBE analog memory records the total time exposure to a given input, regardless of the underlying induction pattern. Cells harboring the circuit shown in Fig. 1C were grown in four different patterns (I to IV) over a 12-day period, where induction by IPTG (1 mM) and aTc (100 ng/mL) is represented by dark gray shading. At the end of each 24-hour incubation period, cells were diluted by 1:1000 into fresh media. The frequency of Kan-resistant cells in the cultures was determined at the end of each day. Dashed lines represent the recombinant allele frequencies predicted by the model (see supplementary materials). Error bars indicate SEM for three independent biological replicates.

  5. Fig. 5 SCRIBE memory operations can be decoupled into independent “input,” “write,” and “read” operations, thus facilitating greater control over addressable memory registers in genomic tape recorders and the creation of sample-and-hold circuits.

    (A) We built a circuit where information about the first inducer (aTc) is recorded in the population, which can then be read later upon addition of a second inducer (IPTG) that triggers a read operation. We created an IPTG-inducible lacZOFF locus in the DH5αPRO background, which contains the full-length lacZ gene with two premature stop codons inside the open-reading frame. Expression of ssDNA(lacZ)ON from the aTc-inducible SCRIBE(lacZ)ON cassette results in the reversion of the stop codons inside lacZOFF to yield the lacZON genotype. (B) Cells harboring the circuit shown in (A) were grown in the presence of different levels of aTc for 24 hours at 30°C to enable recording into genomic DNA. Subsequently, cell populations were diluted into fresh media without or with IPTG (1 mM) and incubated at 37°C for 8 hours. (C) Total LacZ activity in these cultures was measured using a fluorogenic lacZ substrate (FDG) assay. The red dashed brackets marked with asterisks connect the closest data points of IPTG-induced samples that are statistically significant (P < 0.05 based on one-tailed Welch’s t test). A.U., arbitrary units. (D) We extended the circuit in (A) to create a sample-and-hold circuit where input, write, and read operations are independently controlled. This feature enables the creation of addressable read/write memory registers in the genomic DNA tape. Induction of cells with the input signal (AHL) produces ssDNA(lacZ)ON, which targets the genomic lacZOFF locus for reversion to the WT sequence. In the presence of the write signal (aTc), which expresses Beta, ssDNA(lacZ)ON is recombined into the lacZOFF locus and produces the lacZON genotype. Thus, the write signal enables the input signal to be sampled and held in memory. The total LacZ activity in the cell populations is retrieved by adding the read signal (IPTG). (E) Cells harboring the circuit shown in (D) were induced with different combinations of aTc (100 ng/ml) and AHL (50 ng/ml) for 24 hours, after which the cultures were diluted in fresh media with or without IPTG (1 mM). These cultures were then incubated at 37°C for 8 hours and assayed for total LacZ activity with the FDG assay. (F) Cell populations that received both the input and write signals followed by the read signal exhibited enhanced levels of total LacZ activity. Error bars indicate SEM for three independent biological replicates.

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