Tokyo/Genetic circuit

From 2007.igem.org

Abstract　　Concept & Model　　Requirements　　Genetic_circuit　　Works　　About_our_team

Genetic Circuit

Fig.1 is an outline of the genetic circuit in our model, designed to realize our Concept & Model.

All cells, wokers and idlers, have THE SAME genetic circuit (just as 'society' members have practically the same genetic backgrounds).

Fig.1: Genetic Circuit
All cells, wokers and idlers, have the same genetic circuit. Workers expresses RFP; while, idlers GFP.

Workers (State A)

As shown in Fig. 2, an A-state cell, in which the promoter Pa is "on," expresses the repressor Ra, LuxI, and RFP. The repressor Ra "turns off" the promoter Pb and inhibits the cell-state change. This cell also expresses LuxI, which makes AHL, cell-cell communication molecules. Downstream of these genes, there is an RFP gene; therefore, we would be able to detect A-state cell using flow cytometer.

Fig.2: Workers (State A)
A worker, an A-state cell expresses the Pa’s downstream genes including the repressor Ra, luxI, and RFP. The Pb and its downstream genes for idlers are positively repressed.

Idlers (State B)

Fig. 3 shows a B-state cell, in which promoter Pb is "turned on" in the presence of AHL, and repressor Rb and GFP are expressed. Repressor Rb represses the expression of promoter Pa and thereby RFP; therefore, fluorescence of GFP is used to detect B-state cells.

If Workers are removed...

As shown in Fig 4, if A-state cells are removed, B-state cells sense the absense of A-cells. Responding to the decrease of AHL, cells stop producing repressor Rb. Now that repressions of Pa by Rb as well as that of Pb by Ra are declined, either states cannot be clearly expressed, as if the cells were reset.

Fig.3: Idlers (State-B)
In the presence of enough AHL, an idler, a B-state cell expresses the Pb’s downstream genes including the repressor Rb and GFP. The Pa and its downstream genes for workers are positively repressed.

Fig.4: If Workers are removed...
If workers are removed, AHL concentration decreases, which declines activation of promoter Pb. With less gene expression downstream of Pb, the B-state becomes unstable.

Hybrid promoter

To realize such a genetic circuit, Pb should be "Hybrid promoter" regulated by AHL and LacI as shown in the figures.

cf. Other suggestions on the genetic circuit on the A side

As to genetic circuits to realize our Concept & Model, there were some other suggestions as fololows:

Suggestion 1.

Merit:
The repressor R1 can be inserted after constructing the other part.

Suggestion 2.

Merit:
A ribosome binding site here is easily exchangeable; therefore, the expression level can be modulated without changing the promoter and regulators.

Suggestion 3.

Merit:
Promoters can be easily exchanged, and the expression level can be modulated at promoter level.

Suggestion 4.

Merit:
The precursor of this part was constructed for the functional check of BioBrick parts and available;
therefore, this construction can be done in much shorter time.