Generation of a large family of genetic logic gates for applications in biosensing and information processing

Electronic logic gates are the building blocks of all the digital devices, such as computers and smart phones, on which we have come to rely. Individual logic gates take one or more digital (ON or OFF) inputs, perform a logical operation and produce a single digital output. For example, the output of an AND gate is ON only if both of its inputs are ON, whereas the output of an OR gate is ON if either (or both) of its inputs is ON. Electronic logic gates can be connected together into circuits that can store data, add and subtract numbers, count, and carry out any other logical or mathematical calculation. The microprocessor at the heart of a typical modern digital computer has tens of millions of logic gates, all connected together into complex circuits.

Scientists working on simple bacterial cells have used them to create genetic networks that can reproduce the behaviour of individual logic gates. Just like their electronic counterparts, these biological logic gates can take one or more inputs, perform a logical operation and provide an output. For example, a bacterial AND gate has been engineered to take its input from two natural sensors in the bacterial cell that detect different sugars in the environment. The logic gate combines these two inputs and produces an output, in the form of a fluorescent protein, only when both sugars are present. The hope is that one day these biological logic gates can be engineered into more complex circuits that can perform multiple logical operations, store information, make complex preprogrammed decisions, and have multiple outputs. For instance, a cell could be engineered to detect pollutants and to take action to neutralize the exact cocktail of pollutants present, or to undergo a complex series of metabolic steps to turn agricultural waste into valuable chemical feedstocks or pharmaceutical products.

Each biological logic gate has to be individually built from genetic components known as transcription factors and promoters. Transcription factors and promoters are used by cells to control their own gene expression in response to their environment. To ensure that individual logic gates operate independently of each other in a circuit with multiple logic gates, a different transcription factor must be used for each logic gate. Up until now, transcription factors and promoters had to be sourced from natural systems and characterised one by one, and the lack of a large number of different, well characterised transcription factors has severely limited the complexity of digital genetic circuits that can be built.

In the research proposed here, we will develop a new way to produce a large set of transcription factors and promoters that do not interfere with each other's action. Unlike other transcription factors that have been used to build genetic logic gates to date, these transcription factors will not cause unwanted side effects by altering gene expression in their bacterial host. Therefore, the set of transcription factors we generate will be ideal for building large genetic circuits, containing multiple logic gates, to carry out complex data processing and storage operations.

Application of biological logic circuits for in-the-field detection of pollutants, toxic agents, pathogens, water contaminants, explosives etc. will depend on miniaturised reliable detectors of their outputs. We will develop a miniature device to detect fluorescent proteins produced as outputs of bacterial genetic logic circuits, and will test this device using a genetic circuit built from our new transcription factors. This should pave the way towards the development of portable, easy to use biological sensors and processors for a whole host of applications.

An important goal in synthetic biology is to build genetic circuits to process and store information. These circuits could take inputs from biosensors to detect pathogens, toxic agents, explosives etc., process these inputs, and take appropriate action. Synthetic gene circuits are based on regulated promoters and transcription factors (TFs) that bind operator sites to regulate transcription. Promoters with binding sites for multiple TFs can carry out combinatorial logic operations, combining several inputs into a single output. Genetic logic gates can be connected together into circuits by using one promoter to control the expression of a TF for another. However, each TF can be used only once in any cell, as reusing them will cause unwanted cross-talk between different parts of the circuit. There are relatively few well characterized TFs that can be used in genetic logic gates. Therefore, to allow the construction of more complex genetic circuits, there is a pressing need for new TFs that do not interfere with each other's action.

We will generate a family of orthogonal zinc finger TFs and regulated promoters to act as combinatorial logic gates. Zinc fingers recognize DNA sequences with high affinity and specificity, and can be engineered to bind to almost any chosen DNA sequence, facilitating the production of a large set of TFs and regulated promoters. These zinc finger logic gates will be used to build a demonstration genetic circuit, with multiple inputs and fluorescent outputs at multiple wavelengths.

To test this synthetic genetic circuit, we will design and fabricate an integrated microfluidics device, consisting of an arrayed waveguide grating coupled to a CCD detector, capable of detecting fluorescence at multiple wavelengths from a single bacterial cell. The small footprint of the device will permit easy integration with additional modules, and suggests a promising route to stand-alone novel biological sensors for field and military applications.

The work proposed here aims to generate a set of transcription factors and regulated promoters that can act as digital logic gates in synthetic genetic circuits. These circuits can be used in a broad range of biological computing and information storage applications, including biosensors, reprogramming bacterial metabolism for biofuel and energy generation, decontaminating land and water and biomanufacturing of pharmaceuticals. The logic gates we produce will provide a synthetic biology toolbox for building biological computing devices of far greater complexity than previously envisioned. We will illustrate the potential of these biological logic gates by building a demonstration genetic circuit with three different fluorescent proteins as outputs, alongside a miniaturised microfluidics platform for the detection of these outputs.

This is an enabling technology that will allow future work in synthetic biology to produce a whole range of new applications. There are therefore a broad range of impacts and beneficiaries including :

i) Academic beneficiaries (as outlined in the academic beneficiaries section above)
ii) Military beneficiaries from the development of new biosensors. For instance, circuits could be engineered into cells to process inputs from separate sensors for solvents, plasticisers and other components of explosives, and to provide an output only if all of these elements were present, providing for robust detection of explosives. The miniaturised microfluidics detector we are building could provide cheap, easy to use, in-the-field biosensors for a range of different compounds.
iii) Industrial and other business beneficiaries. For instance, genetic circuits could be used to re-programme bacterial cells used for biotechnology applications so that they respond to different feedstocks, making the most appropriate use of the nutrients available. This would lead to more efficient conversion of feedstocks into valuable products such as biofuels, pharmaceuticals or food supplements. In a related approach, a digital event counter within a cell could be used to drive progression through a program of different patterns of gene expression necessary for the different steps in an industrial fermentation process, much like a washing machine progressing through wash, rinse and spin cycles.
iv) Environmental benefits. Synthetic genetic circuits could be built into bacteria or other organisms engineered to remove pollutants from the environment. Multiple biosensors could detect low levels of pollutants and direct the organism to move up the concentration gradient towards the source of the pollution. Once the pollutants reach a high enough level, metabolic pathways could be switched on to degrade only those pollutants that are present.