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Permanent genetic memory with >1-byte capacity

Abstract

Genetic memory enables the recording of information in the DNA of living cells. Memory can record a transient environmental signal or cell state that is then recalled at a later time. Permanent memory is implemented using irreversible recombinases that invert the orientation of a unit of DNA, corresponding to the [0,1] state of a bit. To expand the memory capacity, we have applied bioinformatics to identify 34 phage integrases (and their cognate attB and attP recognition sites), from which we build 11 memory switches that are perfectly orthogonal to each other and the FimE and HbiF bacterial invertases. Using these switches, a memory array is constructed in Escherichia coli that can record 1.375 bytes of information. It is demonstrated that the recombinases can be layered and used to permanently record the transient state of a transcriptional logic gate.

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Figure 1: Discovery of phage recombinases and their recognition sites.
Figure 2: Memory switch characterization.
Figure 3: Incorporation of recombinases into larger genetic circuits.

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Acknowledgements

C.A.V., T.K.L. and L.Y. are supported by the US Defense Advanced Research Projects Agency (DARPA CLIO N66001-12-C-4016). C.A.V. and A.A.K.N. are supported by DARPA CLIO N66001-12-C-4018. C.A.V., M.T.L., A.A.K.N. and J.F.-R. are supported by the Office of Naval Research Multidisciplinary University Research Initiative (N00014-13-1-0074; Boston University MURI award 4500000552). C.A.V. is also supported by US National Institutes of Health (GM095765), the US National Institute of General Medical Sciences (P50 GMO98792) and the US National Science Foundation Synthetic Biology Engineering Research Center (SynBERC EEC0540879). A.A.K.N. receives government support FA9550-11-C-0028 and is supported by the National Defense Science and Engineering Graduate Fellowship 32 CFR 168a from the US Department of Defense Air Force Office of Scientific Research.

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Authors

Contributions

C.A.V. and L.Y. conceived of the study and designed the experiments. L.Y., A.A.K.N., C.J.M. and J.F.-R. performed the experiments and analyzed the data. C.A.V., L.Y., A.A.K.N. and C.J.M. wrote the manuscript. C.A.V., T.K.L. and M.T.L. managed the project.

Corresponding author

Correspondence to Christopher A Voigt.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The alignment used to identify the core sequence from a conserved protein split by a prophage.

The yellow line indicates the prophage genome and blue lines indicate the regions in bacterial genome that flank the prophage.

Supplementary Figure 2 The phylogenetic tree for the complete set of 34 integrases.

The Genbank ID of the integrases and the attB/P sequences are provided in Supplementary Table 1. The < symbols mark the 13 integrases used to build memory switches.

Supplementary Figure 3 Additional data corresponding to the memory switch characterization.

The data corresponds to the switches presented in Figure 2. (A) The geometric average of GFP fluorescence measured at the uninduced state (0mM arabinose, 0.5% glucose, blank, –) and induced (1mM arabinose, grey, +) states is shown. (B) The fractions of the GFP ON cell population at uninduced (blank, -) and induced (grey, +) state are listed. Asterisks indicate values lower than 0.5%. The ON population is defined as having fluorescence values between 102 and 105 au. The average and standard deviation were calculated from three independent experiments performed on different days. (C) PCR bands amplified from cell cultures before (–) and after (+) arabinose induction using the primer 1 and 2 shown in part a. The expected band size is 0.8 kb. (D) The fraction of cells that are ON is shown versus time. Cells are induced at t = 0. The average and standard deviation is shown for three independent experiments.

Supplementary Figure 4 The attP site for Int13 has constitutive promoter activity.

Prior to flipping, GFP is expressed (grey line). By swapping the attP and attB locations, the expression is eliminated (black line). This data is for E. coli DH10b containing only the reporter plasmid (no integrase plasmid) in LB media supplemented with 34 mg/ml Cm grown for 16 hours. The dashed line marks the threshold of 102 used to differentiate between OFF and ON cells. The figure represents three independent replicates.

Supplementary Figure 5 Cytometry data corresponding to the orthogonality matrix.

Cytometry data corresponding to the orthogonality matrix in Figure 2e is shown. Cognate interactions between recombinases and recognition sites are shown on the diagonal in black. Off-target combinations are shown in grey. The dashed line at 102 a.u. shows the threshold used to differentiate on and off cells. This figure represents three experiments conducted on different days.

Supplementary Figure 6 Orthogonality matrix shown in GFP fluorescence

Orthogonality matrix of recombinases and their recognition sites shown in GFP fluorescence corresponding to Figure 2e is demonstrated. The data represent the average of three biological replicates conducted in different days. The average values and standard deviations are shown in Supplementary Table 6.

Supplementary Figure 7 The function and sensitivity of the memory array.

At the induced state [2mM arabinose] the memory array was able to genetically record the expression of each recombines independently (same as Figure 3b diagonal bands). At the uninduced state (0mM arabinose, 0.4% glucose) there is negligible background switching.

Supplementary Figure 8 Fluorescence histograms corresponding to the [1,1] input state for the genetic circuit time courses in Figure 3d.

Inducers were added directly after the 0 hr timepoint, and removed after the 7 h timepoint. (A) Population fluorescence timecourse for the transcriptional AND gate driving YFP, without integrase. (B) Population fluorescence timecourse for the transcriptional AND gate driving Int2, causing the GFP gene to be flipped and expressed.

Supplementary Figure 9 The two-integrase cascade.

(A) A schematic of the recombinase cascade is shown. The plasmid map is shown in Supplementary Figure 13 (pCas_5+7_gfp and pInt5). (B) The cells were uninduced (white) or induced with 1mM arabinose for 8 h (grey). The dashed line at 102 a.u. shows the threshold used to differentiate ON and OFF cells.

Supplementary Figure 10 The impact of single- or multiple-integrase on cell growth.

Cells containing genetic circuits of single (Int2, Int5, Int7 or Int8) or multiple (Int2/5, Int2/5/7 or Int 2/5/7/8) were compared at different concentrations of arabinose. The cells were incubated in at 37 °C for 12 hours with various concentrations of arabinose. The final OD (600nm) decreased slightly in cells containing single integrases at high concentration of arabinose for cells expressing single integrases. However we did not observe additive effects when multiple integrases are incorporated, suggesting that using multiple integrases in parallel is not constrained by growth defect. The average and standard deviations of three independent experiments is shown. Yellow, pInt2; Red, Int5; Blue, Int7; Green, Int8; White, Cis_2+5+7+8; Light grey, Cas_2+5; Dark grey, Cas_2+5+7.

Supplementary Figure 11 Plasmids used to construct AND gate.

The plasmids pAND_Int2 and pAND_reporter were used to conduct memory logic. The plasmid pAND-yfp and pSpec (containing PTet-mRFP1 which was unused in these experiments) were used to generate an AND gate without memory (output YFP). Genetic parts are labeled above or below construct. Promoters are indicated with a bent arrow, genes with a thick arrow, insulators with a circle and a dashed line, RBSs with a solid half-circle and terminators with a T.

Supplementary Figure 12 Plasmids used to construct cascade of integrases.

The 2-integrase cascade of Supplementary Figure 9 was constructed using pInt5 and pCas_5+7_gfp. The 3-integrase cascade of Figure 3e and f was built with pCas_2+5 and pCas_5+7_gfp. Genetic parts are labeled above or below construct. Promoters are indicated with a bent arrow, genes with a thick arrow, insulators with a circle and a dashed line, RBSs with a solid half-circle and terminators with a T.

Supplementary Figure 13 Plasmids used to write 2, 3 or 4 bits of information on the memory array.

pCis_2+5, pCis_7+8, pCis_7+8+10 and pCis_2+7+8+5 were used to write 2, 2, 3, and 4 bits information on the memory array, respectively (Fig. 3g). These plasmids and the memory array plasmid (Fig. 3a) were used for Figure 3g. Genetic parts are labeled above or below construct. Promoters are indicated with a bent arrow, genes with a thick arrow, insulators with a circle and a dashed line, RBSs with a solid half-circle and terminators with a T.

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Supplementary Figures 1–13, Supplementary Tables 1–9 and Supplementary Notes 1–3 (PDF 2229 kb)

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Yang, L., Nielsen, A., Fernandez-Rodriguez, J. et al. Permanent genetic memory with >1-byte capacity. Nat Methods 11, 1261–1266 (2014). https://doi.org/10.1038/nmeth.3147

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