Team:Tsinghua-A/fluid medium 1
TSINGHUA-A
I Introduction
As stated in the orthogonality test in experiment part, we failed to find the orthometric gene circuit of two warriors. We could only find an orthometric gene circuit for one warrior. That is, we can put LuxI, RhlR and Prhl inside the warrior II, and let warrior I secrete C4HSL. From the experiment results, we know that warrior II just response to C4HSL secreted by warrior 1, but not 3OC6HSL secreted by himself.
Discripton1
However, it is hard to determine which receptor and promoter should be put into warrior I. Since from the experiment results, we can see once the receptor-promoter combination response to 3OC6HSL, it will also response to C4HSL. [Fig. 2]
Discripton2
Therefore, what could we do to construct an orthometric gene circuit?
II Design of orthometric gene circuit
The problem now becomes how to block warrior II's response to C4HSL secreted by himself. An intuitive answer to this question is that we could let C4HSL inside warrior I specifically stimulate the expression of an inhibitor, and then the inhibitor can inhibit the promoter of LacI.
Considering this design principle, we design the gene circuit of warrior I and warrior II as follows. [Fig. 3]
Considering this design principle, we design the gene circuit of warrior I and warrior II as follows. [Fig. 3]
Discripton3
Combining with the experiment data [Fig. 2], we can find C4HSL can specifically bind to RhlR and then the complex bind to Prhl so that TetR can be expressed. Then TetR can bind to Plux/tet and inhibit the expression of LacI.
By this circuit, we can see that even though C4HSL-RhlR can stimulate expression of LacI by binding to Plux/tet directly, its stimulation of TetR at the same time can counteract this effect and so warrior 1 won't response to C4HSL secreted by himself.
By this circuit, we can see that even though C4HSL-RhlR can stimulate expression of LacI by binding to Plux/tet directly, its stimulation of TetR at the same time can counteract this effect and so warrior 1 won't response to C4HSL secreted by himself.
III Evaluation of orthogonality by model
Like in the orthogonality test of experiment part, gene circuit of two warriors is orthometric when the AHL molecules secreted by warrior I can just activate the promoter inside warrior II, but cannot activate the promoter inside itself and vice versa. Also, we use RFP intensity to indicate the level of LacI inside cell.
Therefore, we can know the orthogonality by looking at the change of warrior I's intensity as time with or without warrior II. (Because warrior II can just respond to C4HSL according to the experiment results, so it is accord with the orthogonality requirement and we do not consider response of warrior II)
Therefore, we can know the orthogonality by looking at the change of warrior I's intensity as time with or without warrior II. (Because warrior II can just respond to C4HSL according to the experiment results, so it is accord with the orthogonality requirement and we do not consider response of warrior II)
IV ODE, parameters and its resource
V Original Parameter set of the model
VI Results of model
1. Results under original parameter sets:
As stated above, RFP intensity is used to indicate the response of the warrior. So from this original parameter set and result, we can see that there is no distinguish between the response of warrior I without with warrior II, so orthogonality is not good. In the real situation, this means that the warrior I has high amount of LacI even without warrior II and thus almost no CmR. So it has no resistance to chloramphenicol and will be killed by itself.
But how could this happen? Intuitively, we may hypothesize that TetR actually not inhibit the expression successfully when its expression is activated by C4HSL-RhlR. So we may solve this problem by increasing the maximal expression rate of TetR. In the experiment, this can be achieved by changing the RBS after Prhl of TetR in warrior I.
But how could this happen? Intuitively, we may hypothesize that TetR actually not inhibit the expression successfully when its expression is activated by C4HSL-RhlR. So we may solve this problem by increasing the maximal expression rate of TetR. In the experiment, this can be achieved by changing the RBS after Prhl of TetR in warrior I.
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(Fig. 4 RFP level inside warrior I with or without warrior II beta_(1AHL_1t )=6e3) |
2. Increasing the maximal expression rate of TetR in warrior I
Change beta_(1AHL_1t ) from 6e3 to 6e5
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From the results of the model, we are delight to see that warrior I just respond when there is warrior II. So the orthogonality of gene circuit is satisfied!
However, if the level of TetR is too high (like when beta1ahl1t=6e7), we will see that warrior I cannot respond with or without warrior II.
However, if the level of TetR is too high (like when beta1ahl1t=6e7), we will see that warrior I cannot respond with or without warrior II.
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In conclusion, the core is that we can make the gene circuit orthometric by regulating the TetR level to a moderate level. This can be easily achieved by change the RBS after Prhl of TetR in warrior I.
According to the results obtained from our model, it is clear what we should do in the experiment to achieve the goal.
According to the results obtained from our model, it is clear what we should do in the experiment to achieve the goal.
VII Reference
[1] Pai A, You L. Optimal tuning of bacterial sensing potential. Mol Syst Biol, 2009, 5:286 https://www.ncbi.nlm.nih.gov/pubmed/19584835
[2] Ron M, Rob P. Cell Biology by the numbers. United States: Garland Science, 2015 http://book.bionumbers.org/
[3] ETH_Zurich 2014: https://2014.igem.org/Team:ETH_Zurich [4] Ye C, Jae K. K, Andrew J. H. Emergent genetic oscillations in a synthetic microbial consortium Science, 2015, 349:6215
[2] Ron M, Rob P. Cell Biology by the numbers. United States: Garland Science, 2015 http://book.bionumbers.org/
[3] ETH_Zurich 2014: https://2014.igem.org/Team:ETH_Zurich [4] Ye C, Jae K. K, Andrew J. H. Emergent genetic oscillations in a synthetic microbial consortium Science, 2015, 349:6215
