Synthetic gene circuits
A characterised collection of constitutive or inducible promoters and of ribosome binding sites, measured in the same host against the same controls, so that a required expression level can be reached by combination rather than by guesswork. Because the strengths multiply, a few dozen combinations cover four or five orders of magnitude, and the practical limit at the top is the host's capacity rather than the parts.
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Promoter / RBS libraries
A characterised collection of constitutive or inducible promoters and of ribosome binding sites, measured in the same host against the same controls, so that a required expression level can be reached by combination rather than by guesswork. Because the strengths multiply, a few dozen combinations cover four or five orders of magnitude, and the practical limit at the top is the host's capacity rather than the parts.
How it differs from the standard method
- Promoter strength and ribosome binding site strength and copy number all multiply; a tuning move is a ratio.
- Part strengths depend on the sequence context, so a catalogue value needs re-measuring in your construct.
- Measured per cell by flow cytometry where possible: a population that has split reads as one intermediate level in bulk.
- The strongest combinations are often the worst performers, because burden selects for cells that have stopped expressing.
1Genetic parts wired into a function
A synthetic gene circuit is a set of genetic parts (promoters, ribosome binding sites, coding sequences, terminators) arranged so that the cell carrying them computes something: senses a molecule and responds, holds a memory of a past event, oscillates, or kills itself when a condition is met. The parts are the same ones molecular biology has used for decades.
Promoter and RBS libraries
A handful of constitutive promoters and a handful of ribosome binding sites, characterised in the same host and measured against the same controls, cover four or five orders of magnitude in combination. This is how a module is matched to the one downstream of it.
Toggle switches
Two repressors, each driving a promoter that represses the other. With cooperativity, matched strengths and low leak the system has two stable states, and a transient inducer pulse moves it from one to the other permanently.
Repressilators and relaxation oscillators
Three repressors in a ring, or a coupled activator and repressor, give sustained oscillations whose period is set by protein lifetimes and delays rather than by anything resembling a clock.
Logic and sensors
Orthogonal repressors layered into NOR gates, riboswitches and two-component systems reading the environment, and recombinase-based circuits that write their state into the DNA.
2Burden: the host is part of the circuit
Expression competes with growth for a shared pool of ribosomes, charged tRNAs and precursors. Push a construct hard and the cell grows measurably slower, and a cell that loses the plasmid, or acquires a mutation that breaks the promoter, grows faster.
3Bistability has three requirements, and they must all hold
For the standard two-repressor toggle, write the steady state of each repressor as its promoter's output under repression by the other. Plotting one against the other gives two nullclines, and the system has as many steady states as the nullclines have crossings: one crossing means one state and no memory, three crossings mean two stable states separated by an unstable one.
4How it goes wrong
Measurement first. Fluorescence units mean nothing unless the detector setting is fixed across every sample and control in the run, and output has to be quoted above the host's own fluorescence, which is a substantial signal.
Key terms
Practise these as flashcards →A module's steady-state output as a function of its input; its shape, not just its range, decides whether a design works.
The steepness of a promoter's response to its regulator; above one for a cooperative repressor, and a prerequisite for bistability.
Output of a fully repressed promoter, as a fraction of its maximum; sets how off the off state can be.
Ratio of a promoter's fully induced to fully repressed output; bounded above by the leak.
The growth cost of expressing a circuit, through competition for the shared translation machinery.
The set of states where one variable is at steady state; crossings of two nullclines are the circuit's steady states.
Two stable steady states for the same conditions, so the circuit's state depends on its history.
Plasmids per cell, set by the origin of replication; multiplies both output and burden.
Plasmid lost at division when there is no active partition system, typically a fraction of a per cent per generation.
Freedom from cross-talk between parts, and between the circuit and the host's own regulation.
Defined spacers or ribozymes that make a part's behaviour independent of the sequence next to it.
When to use it
- Matching one module's expression level to what the next one needs, which is a measurement and tuning problem rather than a cloning one.
- Building a cellular memory, so that a transient exposure leaves a permanent, heritable record.
- Making a biosensor whose output is a reporter, a growth phenotype or a colour change in response to a defined input.
- Engineering a therapeutic cell to act only under a defined condition: a receptor, a logic gate, a safety switch.
- Testing a quantitative model of regulation, by building the circuit the model predicts and measuring whether it behaves as predicted.
Compared with alternatives
The measurement underneath all of this: what a promoter's output actually is, and how it is calibrated.
Per-cell measurement, which is what distinguishes a split population from a uniformly intermediate one.
How a multi-part construct is physically built, and why defined junctions matter for part behaviour.
When a part or a circuit cannot be designed to the required behaviour, it can sometimes be selected for instead.
Its CRISPRi and CRISPRa arms use the programmable repressor and activator that circuits borrow as parts, which is where many orthogonal regulators come from.
How circuits are delivered to mammalian cells, including CAR constructs, and what copy number that gives.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A characterised collection of constitutive or inducible promoters and of ribosome binding sites, measured in the same host against the same controls, so that a required expression level can be reached by combination rather than by guesswork. Because the strengths multiply, a few dozen combinations cover four or five orders of magnitude, and the practical limit at the top is the host's capacity rather than the parts.
- Promoter strength and ribosome binding site strength and copy number all multiply; a tuning move is a ratio.
- Part strengths depend on the sequence context, so a catalogue value needs re-measuring in your construct.
- Measured per cell by flow cytometry where possible: a population that has split reads as one intermediate level in bulk.
- The strongest combinations are often the worst performers, because burden selects for cells that have stopped expressing.