In our system, we have three characters. Warriors attack the enemies, farmers provide nutrients for everybody, while beggars sit there doing nothing.
    To contruct our farmers, we contructed a new part invertase(BBa_K2250000/BBa_K2250001/BBa_K2250003). Our E.coli MG1655 can not utilize sucrose as its nutrients. Invertase catalyzes sucrose into glucose and fructose, thus providing nutrients for all bacteria. See the intervase test for more details.

    To realize the role of the warrior, we utilized a kind of molecule called AHL([N-]acyl-homoserine lactones). There are several kinds of AHLs, which bind to different receptors with different affinity. AHL together with the receptor can bind to specific promoters and activate the corresponding gene. We want to design a gene circuit with orthogonality.In other words, we want the warrior to kill bacteria from the other group but not from its own group. To accomplish this, we did the orthogonality test to select the comparatively best pairs.
    After the construction of our warriors, we tested how the warriors really work. We have two standards to see if the warriors are fitted or not. First, the warrior must have good orthogonality. That is to say, the warrior must kill its enemies but not itself. Second, the killing ability must be neither too strong nor too weak. We carried out the killing test to test our warriors.

    From the killing test, we find that although warrior 2 is nearly perfect, but warrior 1 kills itself and is too weak. We modified the gene circuit in order to enhance the performance of warrior 1. We did this through changing promoters and adding the part TetR. We built a model to help us modify the gene circuit, see improved gene circuit. Apart from this, we a more thorough discussion on killing ability in model part ---regulation of killing ability.

    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. Figure 1 shows the number of living cells in each group. Figure 2 shows the number of cells expressing mRFP in each group. Figure 3 shows the ratio of the number of cells expressing mRFP to the number of living cells.

    Figure 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 Figure 1 and Figure 3 are also in line with expectation. Figure 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.