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

Abstract

Cellular memory is crucial to many natural biological processes and sophisticated synthetic biology applications. Existing cellular memories rely on epigenetic switches or recombinases, which are limited in scalability and recording capacity. In this work, we use the DNA of living cell populations as genomic "tape recorders" for the analog and distributed recording of long-term event histories. We describe a platform for generating single-stranded DNA (ssDNA) in vivo in response to arbitrary transcriptional signals. When coexpressed with a recombinase, these intracellularly expressed ssDNAs target specific genomic DNA addresses, resulting in precise mutations that accumulate in cell populations as a function of the magnitude and duration of the inputs. This platform could enable long-term cellular recorders for environmental and biomedical applications, biological state machines, and enhanced genome engineering strategies.

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

(A) Synthetic ssDNA (red line) 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-value < 0.05 based on one-tailed Welch’s t-test). Error bars indicate the standard error of the mean for three independent biological replicates.

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) galK_ON cells harboring the circuits shown in A) were induced with either IPTG (1 mM) or aTc (100 ng/ml) for 24 hours and the galK_OFF frequencies in the population were determined by plating the cells on appropriate selective conditions. (C) galK_OFF cells (obtained from the experiment described in B)) were induced with IPTG (1 mM) or aTc (100 ng/ml) for 24 hours and the galK_ON frequencies in the population were determined by plating the cells on appropriate selective conditions. Error bars indicate the standard error of the mean for three independent biological replicates.

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

(A) Constructs used to target genomic kanR_OFF and galK_ON loci with IPTG-inducible and aTc-inducible SCRIBE cassettes, respectively. (B) Induction of kanR_OFF galK_ON cells with IPTG or aTc generates cells with the kanR_ON galK_ON or kanR_OFF galK_OFF genotypes, respectively. Induction of kanR_OFF galK_ON cells with IPTG and aTc generates cells with the kanR_ON galK_OFF genotype. (C) kanR_OFF galK_ON reporter cells containing the circuits in A) were induced with different combinations of IPTG (1 mM) and aTc (100 ng/ml) for 24 h 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 the standard error of the mean for three independent biological replicates.

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 (P_Dawn). The yf1/fixJ synthetic operon was expressed from a constitutive promoter – its products cooperatively activate the P_fixK2 promoter, which drives lambda 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 kanR_OFF to kanR_ON. Cells harboring this circuit were grown overnight at 37°C in the dark, diluted 1:1000, and then incubated for 24 h 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 h 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-IV) over a twelve-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 h incubation period, cells were diluted by 1:1000 into fresh media. The number 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 the standard error of the mean for three independent biological replicates.

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 lacZ_OFF 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 lacZ_OFF to yield the lacZ_ON genotype. (B) Cells harboring the circuit shown in A) were grown in the presence of different levels of aTc for 24 h 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-value < 0.05 based on one-tailed Welch’s t-test). (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 lacZ_OFF locus for reversion to the wild-type sequence. In the presence of the “Write” signal (aTc), which expresses Beta, ssDNA(lacZ)_ON is recombined into the lacZ_OFF locus and produces the lacZ_ON 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 h, 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 the standard error of the mean for three independent biological replicates.