Overview
Criticality detection system, which we intend to construct, combines biosensor with signal converter. The work pattern is that the system receives the specific input signal and then generates pulse output when the input’s vibration exceeds predefined threshold (see Fig.1).
Overview
The detection of threshold is achieved with the help of a red-light-sensing part and a specially designed negative feedback circuit. A fusion protein called Cph8 senses red light and activates three OmpC promoters when red light intensity in the environment drops below a threshold. An output (GFP) keeps being generated until it is inhibited by the negative feedback circuit we built (see Fig.2). A relatively long feedback circuit was designed to generate pulse of good intensity and modularity.
Considering the system construction and work pattern, our criticality detection system can function as a pulse generator, a signal filter and so on. In our case, the module is proved to have potential application in biocomputer development and health care.
Our system can be used as a signal filter in the biocomputer development. Combining criticality detection system with pairs of integrase and excisionase, we set up a binary counting system composed of some two-state latch modules that switch between states “0” and “1”.
Working as a pulse generator in dental caries prevention, the system release a suitable amount of antimicrobial peptides by adjusting the length and intensity of the pulse and maintain the population of oral flora at a satisfactory level.
Criticality Detector
The criticality detector could be divided into two parts, a series of protein for light-sensing and a negative feedback for pulse generating.
The light-sensing protein we use is Cph8, a chimeric protein which produces strong response to light. It consist of a light-sensing domain Cph1 and an EnvZ domain.
Phococyanobilin is necessary for Cph1 to response to light, but it’s not naturally produced in E.Coli. Another two genes , ho1 and pcyA, can produce two enzymes that convert the endogenous haem into phococyanobilin. Ho1 is one member of the heme oxygenases family. It functions in producing BV IXa from endogenous heme in E. Coli. Ho1 catalyses stereospecific cleavage of heme and releases Fe2+ and carbon monoxide, which is the first step of phycocyanobilin synthesis. The second step is conducted by a phycocyanobilin: ferredoxin oxidoreductase (pcyA) which functions in reducing BV IXa.
Functioning as a kinase, the EnvZ domain could lead to autophosphorylation of an endogenous regulator OmpR when light intensity remains below threshold. Those OmpR activate the ompC promoter. But when light intensity is high, autophosphorylation is inhibited and therefore, the expression stops(See Fig.3).
Criticality Detector
A negative feedback circuit is constructed to shut down output while maintain it for a while, which generates a suitable pulse output. Three OmpC promoters are set to receive input. The first one controls the transcription of a CI protein with the RBS locked in a cr loop of a artificial riboregulator system. The second one controls the transcription of a taRNA which unlocks the cr loop and starts the expression of CI. The last one expresses GFP before which is a CI binding site. When CI binds to it, GFP output would be shut down and hence generate a pulse(See Fig.4).
Criticality Detector
>The different work patterns of the negative circuit in states of reaching the threshold and after reaching the threshold for a while are showed in Fig.5 and Fig.6 below.
