Team:KULeuven/Project/Memory

Components
BBa_C0052 is the cI repressor from phage 434, modified with an LVA-tag. It is placed under control of BBa_R0053, the phage P22 cII regulated promoter. BBa_C0053 codes for this cII P22 repressor protein and is placed under control of a standardized right cI 434 promoter BBa_R1052. A third subdivision consists of the BBa_C0056 cI repressor from phage 434 under control of the TetR promoter BBa_R0040. In this instance however, the cI 434 protein is not LVA-tagged. The fourth and last subdivision is an antisense LuxI RNA (encoded by BBa_K145013) once again under control of the standardized right cI 434 promoter BBa_R1052.

Action


The memory device works as follows. When there is no signal from the input device (through the TetR promoter), and there has never been one, the P22 c2 repressor will gain control over the system. P22 c2 and not 434 cI controls this OFF state of the memory because it has a lower Kd, a stronger promoter (BBa_R1052 vs BBa_R0053) to fire its transcription and a more efficient RBS for translation (BBa_B0031 vs BBa_B0033) than cI 434. This means that the synthesis of cI 434 (LVA) mRNA will be rapidly and strongly repressed from the c2 P22 regulated promoter. In the mean time, transcription can proceed freely from the cI 434 promoter when this repressor is absent/very low, thus continuing c2 P22 synthesis as well as antisense LuxI RNA from the same promoter, further imposing the OFF state of the memory on the system. The antisense LuxI will disrupt LuxI translation, inhibiting an untimely timer causing untimely cell death.



When an input signal emerges, transcript for the long-lived 434 cI repressor, without the LVA-tag, will be made from the input TetR promoter. The cI 434 produced will effectively shut down transcription of c2 P22 repressor from the cI 434 promoter, ultimately releasing the c2 P22 promoter in front of cI 434 from repression. In this way, cI 434 (with and without LVA) takes over control of the memory and a switch occurs. The amount of cI 434 produced in a pulse of about 1300 seconds is enough to sustain a full switch so that even when the input signal fades away after the short pulse, cI 434 (LVA) will be indefinitely produced from the c2 P22 promoter, repressing transcription of c2 P22 from the cI 434 promoter. Thus the memory has switched to and will remain in the ON state with high cI 434 (LVA) and no c2 P22.

This process is nicely pictured on the modeling page of the memory system.