Team:Tsinghua-A/design of characters
TSINGHUA-A
Design of characters
I Design 1.0
Fig. 1 Design 1.0 of gene circuit
1. How warriors kill enemies:
AHL1 in warrior I excretes AHL1. AHL1 goes into its enemies and forms a complex with the protein AHL1R. This complex activates the promoter Pahl1, thus activating the expression of LacI. The expression of LacI inhibits the promoter Plac, thus inhibiting the expression of cmR. Because we culture our E. coli in medium containing chloramphenicol, warrior I kills its enemies. As AHL1 cannot activate Pahl2 via AHL2R inside E. coli from warrior I’s group, we fulfill our desire that warrior I just kill the enemies but not bacteria from it own group. Warrior II works in a similar way.
2. How farmers provide nutrients:
RafD encodes invertase, which can hydrolyze sucrose into glucose and fructose. The signal peptide of HlyA ligated to RafD can help the invertase be secreted through the recognition of its signal sequence. This is our new part. Besides, by combining extrinsic HlyB, HlyD with intrinsic TolC from the nucleoid in E. coli, we form the transporter which helps the invertase to be secreted.
This whole part is designed to produce a secretory invertase. As our E. coli can use glucose but not sucrose as its carbon source, farmers fulfill the role of providing nutrients.
However, from our Orthogonality test and Killing test, we find that we can just find warrior II that can be killed by warriors from the other group but not itself. Warrior I cannot satisfy this. Therefore, we improved our gene circuit by choosing specific combinations of AHL, receptor and promoter.
AHL1 in warrior I excretes AHL1. AHL1 goes into its enemies and forms a complex with the protein AHL1R. This complex activates the promoter Pahl1, thus activating the expression of LacI. The expression of LacI inhibits the promoter Plac, thus inhibiting the expression of cmR. Because we culture our E. coli in medium containing chloramphenicol, warrior I kills its enemies. As AHL1 cannot activate Pahl2 via AHL2R inside E. coli from warrior I’s group, we fulfill our desire that warrior I just kill the enemies but not bacteria from it own group. Warrior II works in a similar way.
2. How farmers provide nutrients:
RafD encodes invertase, which can hydrolyze sucrose into glucose and fructose. The signal peptide of HlyA ligated to RafD can help the invertase be secreted through the recognition of its signal sequence. This is our new part. Besides, by combining extrinsic HlyB, HlyD with intrinsic TolC from the nucleoid in E. coli, we form the transporter which helps the invertase to be secreted.
This whole part is designed to produce a secretory invertase. As our E. coli can use glucose but not sucrose as its carbon source, farmers fulfill the role of providing nutrients.
However, from our Orthogonality test and Killing test, we find that we can just find warrior II that can be killed by warriors from the other group but not itself. Warrior I cannot satisfy this. Therefore, we improved our gene circuit by choosing specific combinations of AHL, receptor and promoter.
II Design 2.0
Fig. 2 Improved gene circuit of two warriors. The one on the top is warrior I while the one below is warrior II. The gene circuits of beggar and farmer are modified in a similar way.
In this circuit, warrior II can only be killed by C4HSL secreted by warrior I, but not 3OC6HSL secreted by itself.
Without TetR, we can see that C4HSL can activate expression of LacI by binding to RhlR and LuxR from the results of Orthogonality test . However, we have TetR here! C4HSL forms a complex with the protein RhlR inside warrior I. This complex activates the expression of TetR. TetR can then inhibit the promoter Plux/tetR, thus counteracting the self-activating effect. In this way, the expression of LacI is not activated so warrior I does not kill itself. We modify the gene circuits of beggar I and farmer I in a similar way so that warrior I no longer kill them. Its feasibility can be seen in Improved gene cicuit.
Without TetR, we can see that C4HSL can activate expression of LacI by binding to RhlR and LuxR from the results of Orthogonality test . However, we have TetR here! C4HSL forms a complex with the protein RhlR inside warrior I. This complex activates the expression of TetR. TetR can then inhibit the promoter Plux/tetR, thus counteracting the self-activating effect. In this way, the expression of LacI is not activated so warrior I does not kill itself. We modify the gene circuits of beggar I and farmer I in a similar way so that warrior I no longer kill them. Its feasibility can be seen in Improved gene cicuit.
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