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Synthetic biology

Synthetic epigenetic memory

Combining an inducible methyltransferase with a methylation-sensitive zinc finger creates an epigenetic memory circuit.

For many years, Albert Jeltsch at the University of Stuttgart in Germany has pondered the idea of using bacterial methylation as a means of information transfer. “I started my career working on bacterial transferases,” says Jeltsch; “we realized that they imprint barcodes on the DNA but we never came up with a good system to use this.” His team's breakthrough came with the realization that these barcodes could be recognized by DNA-binding zinc finger (ZF) proteins to create cellular memory.

Johannes Maier, a PhD student in the lab, started with a circuit consisting of an engineered ZF that binds a motif in the promoter of an operon encoding the methyltransferase CcrM and the reporter GFP, thereby repressing their transcription. Upon methylation of this motif, the ZF no longer recognizes the site, repression is lifted, and CcrM and GFP are transcribed.

The team then used this basic idea to build more complex systems that could store the memories of conditions the bacteria had encountered. Their first circuit recorded a change in temperature from 30 °C to 37 °C during the culture of Escherichia coli cells. This shift weakened ZF binding at the CcrM promoter and thus ended the repression and triggered the expression of the methyltransferase and the reporter. GFP marked only the cells that had experienced the higher temperatures.

To sense arabinose, Maier used a two-plasmid circuit. One plasmid encoded CcrM from a promoter inducible with arabinose; the other harbored a promoter with a methylation-sensitive motif that was bound by a ZF, which in turn repressed expression of a downstream GFP. Once CcrM expression was triggered by the presence of arabinose, the enzyme methylated the promoter, which removed the repressing ZF and allowed GFP to be expressed.

A memory circuit sensing the presence of arabinose. Credit: Figure adapted from Maier et al., Nature Publishing Group.

The researchers set up similar memory systems for E. coli cells in which a toxin or UV radiation induced CcrM expression. To create a system that could be reset, they fused a degradation signal to CcrM and showed that methylation could be switched on and off, which resulted in oscillating levels of reporter.

Jeltsch sees the power of the method in its ability to multiplex. “Our system is orthogonal;” he explains, “we can write many signals in the genome and detect chemicals of different kinds.” All it will take is to combine engineered ZFs that recognize different motifs, which in turn are methylated by different enzymes that are induced by different inputs. After methylation and derepression the reporters are evidence that the cells encountered toxins.

The team is working on optimizing the system to create logic gates to, for example, trigger methylation only if two input signals are seen. They also attempted to make a detector for radioactivity. “It partially worked,” says Jeltsch, “but the sensitivity was not very good because bacteria are not as sensitive to radiation as one would like them to be for this application.” But he is confident that with a bit of adjustment the radiation sensor will be successful.

Another exciting application for this system is the detection of biomarkers. Jeltsch envisions engineered bacteria that collect methylation signals as they encounter metabolites or tumor markers on their way through the human gut. Evidence of reporters or sequencing of the methylation motifs would show whether the biomarkers are present, and this could help with diagnosis.

References

  1. Maier, J.A.H. et al. Design of synthetic epigenetic circuits featuring memory effects and reversible switiching based on DNA methylation. Nat. Commun. 8, 15336 (2017).

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Rusk, N. Synthetic epigenetic memory. Nat Methods 14, 764 (2017). https://doi.org/10.1038/nmeth.4382

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