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 does not form a complex with protein AHL2R inside E.coli from warrior I’s group, thus we fulfill our desire that warrior I just kill the enemies but not bacteria from it own group. Warrior Il 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, combining with extrinsic HlyB, HlyD and intrinsic TolC from the nucleoid in E.coli together 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.

    In this circuit, warrior II can only be killed by C4HSL secreted by warrior I, but not 3OC6HSL secreted by itself. (This can be shown in orthogonality test )
    Without TetR, we can see that C4HSL can activate expression of LacI by binding to RhlR and LuxR from results of orthogonality test . However, we have TetR here! C4HSL forms a complex with the protein RhlR inside warrior I. This complex activate 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. It feasibility can be seen in Improved gene cicuit.

    RafD, which is primitively from the raffinose operon of E. coli, can express a β-fructofuranosidase. It can hydrolyze raffinose into fructose and meliose, and sucrose into glucose and fructose. HlyA is a secretory protein in bacteria, which can be secreted through the recognition of its signal sequence at the C terminal by a transporter constructed by HlyB, HlyD and TolC. This part is designed to produce a secretory invertase, which can hydrolyze sucrose in media to feed the bacteria without invertase

    As is demonstrated in the genetic circuit, RafD is ligated to the signal peptide of HlyA. The plasmid can also express HlyB and HlyD, but the expression is controlled by PBAD. Besides, TolC originally exists in the genome of E.coli. Thus, the RafD enzyme is able to be transported to the media and hydrolyze the sucrose outside the cells.
    Because the promoter of HlyB and HlyD requires arabinose to work while arabinose is a carbon source, an experiment to test whether this part or the arabinose added to activate the PBAD is what bacteria live on becomes necessary. We used two test parts, secretory invertase and Pcon_mRFP_TT. Each of them are transformed into E. coli MG1655 ΔsidA ΔlacI. E. coli MG1655ΔsidA ΔlacI with different plasmids are called Peasant and Civilian (Despite their similarity to Farmer and Beggar, we cannot call them in that way because their genetic circuits are different after all.) separately, which do not have any invertase genes in genome. Peasant and Civilian will grow in M9-sucrose culture media, a special M9 culture media in which glucose is replaced by sucrose. Then there are only two available carbon sources, sucrose, if hydrolyzed, and arabinose, if added.

    1. Cultivate a pipe of Peasants and a pipe of Civilians for 12h.

    2. Adjust their OD600 to be the same value.

    3. Take 2ml each, and centrifuged them at 12000rpm for 1min. The supernatant was removed and each sediment was resuspended with 2ml M9-sucrose culture media.

    4. Add 5ml M9-sucrose media and 100μl bacterial liquid, in which Peasants and Civilians are mixed, so the total number of them is fixed, nevertheless of their ratio. The concentration of Arabinose was set by adding concentrated Arabinose solution.
The experimental groups and control groups are listed as follows. The number “1” and “3” in the boxes are the numbers of repeats.

    5. All of the groups were cultured for 20h. The growth was measured by flow cytometry. The number of cells is averaged.

    The number of cells were counted and illustrated in the figures. Fig. 1 shows the number of living cells in each group. Fig. 2 shows the number of cells expressing mRFP in each group. Fig. 3 shows the ratio of the number of cells expressing mRFP to the number of living cells.

    Fig. 2 tells us that when Peasants and Civilians coexist in the media, Civilians grow more than the group in which Civilians live alone in the media. Also, the number of Civilians becomes larger than the negative control group if the primitive fraction of peasant is larger. Contrarily, the number of Civilians does not become larger if the primitive quantity of Peasants is too big while the original number of Civilians is too small. And this is considered acceptable. Besides, as is revealed, Civilians grows fastest when the primitive ratio of Peasants and Civilians is 1.
    The results in Fig. 1 and Fig. 3 are also in line with expectation. Fig. 1 demonstrates that when the concentration of Arabinose is 40μM, Civilians cannot live without Peasants, which means the cells in the media mainly live on sucrose instead of arabinose. However, Civilians can grow alone if the concentration of arabinose is up to 100 μM. This comparison tells that 40μM is an appropriate concentration which is able to ask cells to live on sucrose but not arabinose.

     In our project, we hope that the warriors only kill the bacteria from the other side, but not his own side. In other words, the orthogonality of the gene circuit must be good. Necessarily, it means that 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. (Note: In this text, we mean the same when we refer to response intensity of AHL promoter and killing effect of warriors, that is to say, if the response intensity of promoter to AHL secreted by warrior A inside warrior B is high---A may equals to B, it means that the warrior A can be effectively killed by warrior B and vice versa.)
    To help us choose appropriate circuit of two warriors, we should know how specific receptor-promoter combination respond to specific AHL.
    This is characterized by ETH_Zurich 2014. However, the way they characterize the crosstalk may cause inconvenience for us to use. Detailed explanation is shown below:
    The response concentration of some receptor-promoter combinations to AHL molecule may be too high but the concentration range that ETH_Zurich considered is limited, however, in real situation, bacteria we used may be able to secrete higher concentration of AHL molecule. What is more, the reverse situation may also happen. In other words, bacteria we used may be unable to secrete so much AHL molecule to activate gene expression. For example, in our project, we want to design two kinds of E.coli which can synthesize two kinds of AHL and respond to AHL secreted by another but not by itself. Therefore, according to results from ETH_Zurich 2014, shown below (Fig. 1), we may design gene circuit of our E.coli as follows. (Fig. 2)

     In conclusion, it is inconvenient for us to choose our gene circuit just according to results from ETH_Zurich 2014 since it does not consider the ability of E.coli synthesizing AHL. Therefore, we designed our experiment as follows, which consider E.coli’s ability to synthesize AHL.

    To test which kind of AHL, receptor and promoter we can choose to keep the gene circuit orthogonal during the time period of our experiment (For example, in our final experiment, we will co-culture six characters in 3ml medium for 48h), we should detect the killing effect of two warriors in real time.
    However, here for simplicity, we used RFP to indicate amount of LacI inside bacteria, so its intensity inside each bacteria in real time can be used as a relative measurement of the response intensity of specific receptor-promoter to specific AHL and the total killing effect of two warriors in real time.
    What is more, we let E.coli secrete and receive AHL itself to mimic real situation of our system approximately.
    Below are detailed plasmid construction design.
    First, we cloned three AHL synthases---luxI, lasI and rhlI to the low copy backbone---pSB3K3. At the same time, we cloned nine receptor-promoter combinations to pSB6A1. The three receptors are luxR, lasR and rhlR while the three promoters are Plux, Plas and Prhl. RFP is attached to the promoters, which is used to detect the response of the promoters to AHL molecules via the receptors (Fig. 3). After that, we cotransform the plasmids containing AHL synthases and plasmids containing receptor-promoter combinations. As a result, we get 27 different combinations.

    1. Transform MG1655ΔlacI ΔsdiA with 27 kinds of combinations of plasmids mentioned above, which will be used in the orthogonality test.

    2. Pick bacterial clones from the petri plate,then shake it overnight in the LB medium (3ml) with 50μg/ml Ampicilin and 30μg/ml Kanamycin at 37℃. For each combination, 4 clones are picked.

    3. Dilute the overnight culture to 1/50 in fresh LB medium (5ml) containing 50μg/ml Ampicilin and 30μg/ml Kanamycin.

    4. Incubate the fresh cultures at 37℃ until OD600 reaches 0.2.

    5. Add 1ml culture obtained from Procedure 4 to 2ml fresh LB medium containing 50μg/ml Ampicilin and 30μg/ml Kanamycin. Incubate the fresh cultures at 37℃.

    6. Take out 200μl cultures obtained from Procedure 5 and measure the fluorescent intensity and OD600 after 17, 20, 24, 34, 38, 44h. (The cultures will not be put back again.) The excitation wavelength is 584nm, emission wavelength is 607nm.

    From results above, how could we design our two warriors? Let’s show for you!
    First, we need choose two kinds of AHL secreted by each warrior, right? Let’s try to make warrior I secrete C4HSL and warrior II secrete 3OC6HSL. Then which receptor-promoter should we put into two warriors to make them just killed by another warrior but not itself. Let’s first try to determine warrior I, so can we find a receptor-promoter combinations that just respond to 3OC6HSL but not C4HSL? Unfortunately, we can’t! So we cannot choose C4HSL and 3OC6HSL to construct two warriors that we want.
    Similar results of other 2 AHL pairs can be obtained by similar analysis.
    However, we do not need to be so frustrated, since we can determine part of two warriors that satisfy our needs of warrior as follows (Fig. 5) and then our model can tell us how to improve this gene circuit based on this circuit. (Improved gene circuit)

     What is more, because we use RFP to indicate level of killing effect of warriors inside cell during orthogonality test, we are not sure about if the results will be the same (namely, if this indication is reasonable) when we use a complete circuit, so we have Killing test to verify it.

Note: The needs of us about warrior can be decomposed to four parts:
     (1)Warrior I can be killed by warrior II
     (2)Warrior I cannot be killed by itself
     (3)Warrior II can be killed by warrior I
     (4)Warrior II cannot be killed by itself
    We can just satisfy 3 of these needs now, like circuit mentioned in Fig. 5. In other words, the best design we can achieve now by no more other genes is to construct one warrior that can be only killed by the other but not two warriors. Of course, circuit in Fig. 5 is not uniqueness for us to achieve 3 of these needs. However, since it can already satisfy our needs to verify the credibility of orthogonality test in killing test and give a better gene circuit based on it. Therefore, during our project, we use it when we mentioned these two needs, not other circuit.