Binary System
Inspired by the bidirectional DNA flipping system illustrated by Jerome Bonnet, etc in 2012[1], we are planning a binary counting system, using the mechanism of serine recombinases. The counting system is expected to record the number of certain stimulation by the direction states of the registers, which are under the control of serine recombinases. The stimulation should be normalized by the criticality detection system to ensure that the stimulation is properly dealt with.
Basic mechanisms
Recombinases used in this system are large serine recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes [2]. For a species of phage, the recombinases consist of a strictly directional integrase (Int), and a recombination directionality factor (RDF), or called excisionase (Xis), which can change the directionality of Int. The Int can specifically flip the DNA between specific sites (attB and attP), and the sites turn into attL and attR. If a promoter is placed between the pair of att sites, the register is formed, which registers by the directionality of the promoter. As flipping in either direction needs different sets of enzymes, the register may take control in the flipping procedure, by which we can make flip in both direction using the same induction (some standardization work certainly needed). Pairs of recombinases have been found in some phages, and the enzymes as well as att sites are different between each other. Based on this, if we stack up a number of recombinase systems like a binary system, in which the two directionality of each register represents 0 or 1 on one digit, and the overflow to the higher digit is performed by specific RNA polymerases which are highly specific for the exact corresponding promoter. T7 RNA polymerase, for example, can specifically start the transduction after T7 Promoter.
How does it count
We chose two pairs of recombinases, from phage Bxb1 and φC31, and intend to build a counting system with two digits. The two digits are linked by T7 RNA polymerase. As in the figure, the initial status is 00. The first stimulation can induce the expression of Bxb1 Int and Xis, which flips the register from L/R state to B/P state, just like the binary digit is switched from 0 to 1. The next stimulation will flip the register back, and express T7 RNA Polymerase, which induces the flip of the next register: attL/R of φC31. This step is like the binary 01 is added to 10. And the same mechanism goes on. If the registers are more, there should be much larger number that the system can count.
Binary System
Future work
We have to work out the intensity and duration under which each flipping system works best. Hopefully the conditions for both directions of one flipping system are similar, or some adjustments should be taken, to standardize the input and to make good use of the criticality system, which helps standardize the input. Meanwhile, the RNA polymerases are usually rather stable, so the degradation of the RNAP should be accelerated.
[1] Bonnet J, Subsoontorn P, Endy D. Rewritable digital data storage in live cells via engineered control of recombination directionality. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(23):8884-8889. doi:10.1073/pnas.1202344109.
[2] Singh S, Rockenbach K, Dedrick RM, VanDemark A, Hatfull GF. Cross-talk between diverse serine integrases. Journal of molecular biology. 2014;426(2):318-331. doi:10.1016/j.jmb.2013.10.013.
