Memory saving circuits (Siuti et al. 2013, Maier et al. 2016)

[Sophlei]

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Synthetic circuits integrating logic and memory in living cells

Piro Siuti, John Yazbek & Timothy K Lu (2013)

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Global summary:

• Strategy for building synthetic logic circuits in single cells with concomitant DNA-encoded memory storage → assembly artificial logic gates with memory devices

• Creation of 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates

• Results: long-term maintenance of memory for at least 90 cell generations the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR

• Creation of two-bit digital-to-analog converters (= translate digital inducer inputs into stable analog gene expression outputs) → useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers

• Applications: implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications

Method:

• chemical-inducer inputs: drive the expression of orthogonal recombinases from inducible promoters

• these recombinases target genetic components for DNA inversion → conditional gene expression

• building of all possible two-input logic functions: assembly of recombinase-targeted promoters, terminators and output gene modules in various orientation → simple user-defined programming of cellular logic with memory

→ translation of desired computational functions into [promoter(s)]- [terminator(s)]-[output] designs, which can be constructed with straightforward Gibson assembly

Memory:

• stable output memory after the inputs are withdrawn → possible to detect the state of computations using PCR even after cell death

• Possible application: create cellular biosensors whose states can be interrogated in a multiplexed fashion using high-throughput sequencing techniques

Possible applications:

• engineered bacteriophages as a form of portable and transmissible memory
• build autonomous cellular circuits that transition cells in a bioprocess through defined cellular phases, such as biomass accumulation followed by biomolecule production and then cell lysis.
• Cellular biosensors containing logic-and-memory circuits based on irreversible recombinases → history of exposure to individual or logical combinations of environmental signals

Follow up:

• Extending this approach to reversible memory devices → construct sequential logic systems with resettable memory or clocked behaviors
• important challenge: ensuring that transitions between states can be tightly controlled

[Sophlei]

Synthetic circuits integrating logic and memory in living cells.pdf

[Sophlei]

Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation

Johannes A.H. Maier , Raphael Möhrle & Albert Jeltsch (2016)

• Article combining synthetic circuit design with epigenetic mechanisms → construction of synthetic epigenetic memory systems in E. coli allowing to sense temporary stimuli and memorize this information for many cell generations

• Many possible applications described at the end, possible to combine with biosensors

Summary:

• Design of a synthetic epigenetic memory systems: DNA methylation sensitive engineered zinc finger proteins → repress a memory operon comprising the CcrM methyltransferase and a reporter

• Nutrients / ultraviolet irradiation / DNA damaging compounds → induces CcrM expression and DNA methylation

• induced on-state = methylation in the operator of the memory operon prevents zinc finger protein binding → positive feedback and permanent activation

• Reversible switching: mf-Lon protease degradable CcrM variant

• Applications in synthetic biology: life biosensors, death switches or induction systems for industrial protein production

• Large variety of bacterial DNA methyltransferases → massive multiplexing of signal storage and logical operations depending on more than one input signal

Methods:

• Bistable memory systems = artificial regulatory networks (store information in form of DNA methylation patterns in a reversible manner)

• Building the memory system: promoter controlled by CcrM methylation → regulates the expression of a EGFP reporter and of the CcrM MTase CcrM MTase methylates its own promoter → positive feedback

• This epigenetic memory systems can exist in two states: → initial off-state: ZnF repressor binds to the promoter region of the ccrM gene and represses its expression → on-state: weak binding of the ZnF repressor → CcrM is expressed and can methylate its own promoter region DNA methylation of the ZnF binding site weakens ZnF binding → positive feedback loop

• Coupling different trigger systems (sensor elements) to this memory device: ability to detect and memorize physical properties (temperature or ultraviolet radiation, the presence of soluble metabolites or toxic agents)

(Then the article details how they built each part)

Results:

• possible to store information in DNA methylation patterns in dividing bacteria throughout many cell divisions
• can function autonomously in E. coli (sensing heat or arabinose) or as an accessory system attached to endogenous circuits (sensing SOS response)

Potential applications:

• Area of biocontainment: after introducing a gene for a toxin into the memory system, which is stably induced after a certain signal is provided
• kill genetically modified bacteria that have escaped from a bioreactor
• terminate a vaccination with live bacteria in case of medical complications
• large scale industrial protein expression (where induction of regulated expression systems is costly and sometimes difficult to control)
• live bacterial biosensors (a biosensor for radioactivity or toxins, for example in form of small encapsulated and buffered biosensor spheres containing live bacteria)
• biosensors for different toxins or pollutants → cost efficient, long-term observation of environmental sites for industrial pollution
• maintenance of cooled supply chains for pharmaceutical drugs, chemicals or agricultural products
• biomedical diagnosis: sensor systems that respond to biomarkers of diseases, for example, tumour markers in the gut, or biosensor pills that travel through a patient and collect their corresponding signal input for readout after excretion

[Sophlei]

ncomms15336.pdf