Using site-specific recombination to create a synthetic binary counting circuit in living cell

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Jia Zhao, Sean Colloms, Susan Rosser

Institute of Molecular, United Kingdom

Biological modules that can implement information processing and storage are a major long-term goal in Synthetic Biology. Large serine phage integrases are ideally suited for the production of bio-computing and memory modules. These integrase proteins carry out directional cut-and-splice reactions on DNA by recognizing sequences known as attP and attB, cutting them at their centres and rejoining them to form two new sites, attL and attR which do not recombine further. However, in the present of recombination directionality factor (RDF), this directionality is reversed so that attL and attR recombine to re-create attP and attB. If two att sites are placed in inverted repeat, recombination flips the orientation of the intervening sequence, and the two orientations can be thought of as representing a single binary digit (0 or 1 / OFF or ON) heritably stored in the DNA. The state of the DNA can be easily detected by physical means or can drive the expression of a reporter gene such as GFP. We are using phage integrases to build modules for a binary counter that function in living cells. A synthetic circuit has been constructed in E. coli and proper expression levels of Integrase and RDF have been established. By first expressing the integrase on its own, and then the integrase and the RDF together, the binary counter module switches repeatedly from 0 to 1, and then from 1 to 0 each time it receives an external signal. By chaining N units together, each using a different phage integrase, a ripple counter can be made that can count to 2^N. Binary genetic counters could be used to record the number of times the cell has been exposed to a specific environmental event, or to step through a programmed cycle of gene expression patterns.