Key Lock Antikey: Design
The key and antikey system perform a subtraction of the blue light signal and the vanillin receptor signal. The result controls the vanillin production. In biological terms, the subtraction translates as the annealing of complementary RNA strands, the key and the antikey. This reaction is favoured over the reaction between the key and the lock leading to vanillin synthesis. In this way we try to perform the subtraction before inducing production of vanillin.
The biological equivalent of a subtraction can only yield a positive number, so one can only subtract a small from a large amount. Because we can only actively produce vanillin, we have to subtract the measured quantity of vanillin from the wanted quantity, or in other words, the amount of antikey produced by the vanillin receptor from the amount of key produced by the blue light sensor.
The mRNA key and lock sequences form a pair of riboregulators. The lock DNA is located directly upstream of the controlled gene’s RBS. It is folded into a stem-loop secondary structure which prevents access to the ribosome and inhibits translation. Keys are expressed from separate genes (in trans) and code for sequences complementary to the lock. Upon annealing they unlock the ‘closed’ stem-loops, thereby exposing the RBS and permitting expression of the gene(s) downstream.
The most efficient key is a combination of the BBa_J23008 and BBa_J23009 keys. They are placed behind the blue light promoter to ensure transcription after blue light irradiation. BBa_B0015 is used as terminator. This brick consists of BBa_B0010 and BBa_B0012 and is most commonly used. One BBa_J23078 lock is placed before sam5 and sam8 genes and another before COMT to guarantee maximal efficiency of the key/lock system. These genes are part of the vanillin production pathway. Therefore, after a blue light activates transcription of the key, vanillin synthesis starts. This also means that any desired vanillin concentration can be adjusted just by altering irradiation intensity.