To simplify and mimic the complex relationships between populations in real ecosystems, we use methods of synthetic biology to construct a more simple system using E.coli. The system involves two groups of E.coli, with each group having three characters. By secreting AHL molecules, warriors attack the enemies but not E.coli from the same group. Farmers provide nutrients for everybody by the secretion of invertase. Beggars sit there doing nothing, growing and waiting to be killed by warriors from the other group.
    Concretely, the gene circuits are designed as follows. 　　    

    Therefore, our main task is to construct three characters of the two groups.
    Our achievements are shown below.

(1) Construct a new part--invertase
    The invertase used by farmers supports other E.coli when sucrose is the sole carbohydrate source.
    In our system, we hope farmers can produce invertase and secrete it outside the cell to help other E.coli survive. To test if invertase can work as we expected, we transformed invertase and its transport system to E.coli and co-cultured it with cells that can express RFP. We found that E.coli that can produce invertase can truly support the survival of other E.coli. (Fig.2)

(2) Do the orthogonality test for gene circuit design
    It helps us construct warriors that can just be killed by warriors from the other group but not from its own group.
     Because of the similarities between AHLs and between their receptors, warriors may be not only killed by the AHL secreted by the warriors from the other side, but also by itself, if the circuits are not well designed. This is a serious problem we faced when choosing the AHL-receptor-promoter pairs. This problem has been discussed by many groups in iGEM, like ETH_Zurich 2014. However, all previous results are obtained by adding AHL artificially to test the respond of specific receptor-promoter combination. In our project, we let E.coli to secrete AHL itself, so different concentrations of AHL in our system may make the previous studies unsuitable for our system here.
    Therefore, we did Orthogonality test to fit our own needs to help us design gene circuits of warriors. Results are shown below.

    However, we failed to choose an orthogonal gene circuit from our results. We can just determine the circuit of warrior II and what AHL warrior I should secrete, as is shown below:

    We cannot design a warrior I that can be killed by warrior II but not killed by itself regardless of whatever receptor-promoter we put inside warrior I.
(More details can be seen at Orthogonality test)

(3) Verify the orthogonality test
    We constructed our warriors and beggars and used the series of tests listed below to test the creditability of our results from the orthogonality test.
    Because we use RFP to indicate the level of LacI inside the cell during Orthogonality test, we are not sure if the results will be the same when we use a complete circuit. Therefore, we designed two warriors and beggars as below to verify results got from Orthogonality test.

    According to results of Orthogonality test, the warrior I will be killed by itself when it is cultivated without warrior II. Furthermore, warrior II doesn’t kill itself but can kill E.coli from another side successfully. All results we got here are consistent with our Orthogonality test! They greatly enhance the reliability of our previous data. (Fig.6)

(4)Improve the gene circuit of warrior I
    According to our results of the orthogonality test and the killing test, we cannot design a warrior I that can be killed by warrior II but not killed by itself regardless of whatever receptor-promoter pair we put inside warrior I. (See more at orthogonality test)
    The problem now becomes how to block warrior I’s response to C4HSL secreted by himself. Our model tells us if we design warrior I as below (Fig.7), we can make it only be killed by warrior II by just regulating the RBS of TetR to an appropriate intensity.

Results are shown below:

Design a game to make the public more interested in synthetic biology.
    Besides the bench work we did, to realize the educational purpose of our project and to make the public know more about synthetic biology, we designed our game---E.coli War!
    When children played this game during our exhibition in China Science and Technology Museum (CSTM), they found many interesting results which can even promote our understanding of this system. (Fig.9) (See more details at Exhibition at National Museum, https://2017.igem.org/Team:Tsinghua-A/GameGame Overview & https://2017.igem.org/Team:Tsinghua-A/Game_Discovery) Furthermore, this can also be helpful for our further research design.

    Except for a few results in Invertase assay (See more details in Invertase assay), all of our results are obtained by 3 biological replicates, so they are credible.
    In conclusion, we designed a very general system to study many complex relationships and also a useful model to describe this system. In the future, it will be convenient to investigate some interesting questions with regard to relationships between populations and individuals by using this system.
    What’s more, we designed two interesting games--- Fluid E.coli War and Solid E.coli War to make the public more interested in synthetic biology. Conversely, their finding of many interesting results and disciplines in the games could also be of great help to our research. By this way, we combine our project to the world perfectly. Through the development and propaganda of our games, our project can influence our world and vice versa!