1.Project planning

At the project planning stage of this year, we took part in a poster session held at the school festival of the University of Tokyo to show our preliminary plans
(See Policy and Practice for more details) (Fig. 2-1-1).

After communicating with the people in business, we realized that we do not know much about economic system. In order to solve this, we thought of making an educational tool of economics by using E. coli, which is familiar to iGEMers.

Our educational tool for economics has three types of E. coli: Bank, Company, and Customer. (Fig. 2-1-2). Like the exchange of money and products in the real economy, we made these three E. coli which exchange Product and Money in the tool’s economic system. Company makes Product, and sells them to Customer. On the other hand, Customer pays Money to buy Product made by Company.

With this mutual action, Customer and Company build interdependent relationship. Additionally, Bank regulates the Money supply in the market. It functions as a central bank like FRB and European Central Bank (ECB). Here, Product and Money were represented by 3OC12HSL and C4HSL, the signaling molecules of the quorum sensing, respectively.

2. Interdependence between Company and Customer

2.1 Molecular Basis of Interdependence

We designed two genetically engineered E. coli, Company and Customer (Fig. 2-1-3), each of which produces its own quorum sensing molecule for interdependence between the two. Since Company and Customer need each other to continue the now-in-state economy, we designed the interdependence between Company and Customer. Company is dependent on Money supplied by Customer. The signaling molecule C4HSL represents Money. On the other hand, Customer is dependent on Product supplied by Company. The signaling molecule 3OC12HSL represents the Product. The detailed design of the circuit is shown in the following sections.

In the presence of C4HSL, which represents Money, Company can produce chloramphenicol-resistance gene product (CmR) and LasI. CmR protects Company from the antibiotic action of chloramphenicol. LasI produces signaling molecule 3OC12HSL, which represents the Product made by Company. If there is not any C4HSL in the medium, Company cannot produce chloramphenicol-resistance gene product. This will lead to the growth inhibition of Company, which represents Company’s bankruptcy.

The basic design of Customer’s circuit is the same as Company. In the presence of 3OC12HSL, which represents the Product, Customer produces CmR and RhlI. CmR prevents Customer from growth inhibition, and RhlI produces C4HSL, which represents Money. If there is not any Product in the market, Customer cannot produce CmR. This leads to the growth inhibition of Customer.

2.2. Native Prhl Promoter does not satisfy
requirement from system analysis

  To actually make the system of Company and Customer, we first simulated the system to see whether it is feasible or not.

From the simulation, we noticed that the a certain strength of the promoters, Prhl and Plux, need to be close in order to promote Company and Customer’s growth. Detailed analysis is described in the Modeling page.

The graph shown in Fig. 2-2-1 shows that the strength levels of Prhl and Plux promoters must be high and balanced to realize the system. If the strength level of Prhl and Plux promoters are in the red area, Company and Customer can help the growth of each other. However, if the strength level is in the blue area, either one cannot grow well.

    To check whether the intensities of Prhl and Plux promoters satisfy the conditions described above, we examined the strength level of Prhl and Plux promoters. As shown in Fig. 2-1-7, the fluorescence intensity of Plux promoter was about 23-fold higher than that of Plux promoter. Although RBS strength modlulation under Plux promoter might compensate inbalance between expressions HSL-synsase expressions under Plux or Prhl, such modulation corresponds to decreased Plux activity which leads the no growth of Customer and Company.  Therefore, the improvement of Prhl promoter’s strengh level became necessary to meet the modeling results.

2.3. The improvement of Prhl promoter

To meet the modeling results, we added three improved C4HSL-dependent promoters with high maximum expression level by combinations of regulatory-protein binding sites (Fig. 2-1-8).

First, we designed a new Lux promoter which has two RhlR binding sites instead of two LuxR binding sites (Prhl(RR): BBa_K1529320) , as tried in a previous paper (Chuang 2009). To evaluate the function of this promoter, we constructed Prhl(RR)-GFP plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl(RR) promoter showed about 20-fold higher in the fluorescence than that of the original Prhl promoter (BBa_R0071)
(See the Experiment page) (Fig. 2-1-9).

However, Prhl(RR) promoter showed a significant leak in the absence of C4HSL(See the Experiment page). High level of leakage is not suitable for the Company-Customer relationship because their interdependency will be broken.

In order to lessen the leak and increase the maximum expression level, we newly designed two promoters, Prhl(LR) (BBa_K1529310) and Prhl(RL) (BBa_K1529300). These promoters have one LuxR binding site and one RhlR binding site. We changed either the upper RhlR binding site of Prhl(RR) promoter to LuxR binding site (Prhl(LR)), or the latter RhlR binding site to Lux binding site (Prhl(RL)).

One of our new promoter, Prhl(RL) improved in its expression level while keeping the low leakage (Fig. 2-1-9 lane 4). GFP under the control of Prhl(RL) promoter showed about 7-fold higher in the fluorescence than that of the original Prhl promoter. The leak was no more than 2-fold high.

Although the other Prhl(LR) promoter showed a higher maximum expression level, it showed a significant leak like Prhl(RR) promoter (Fig. 2-1-9 lane 3). GFP under the control of Prhl(LR) promoter showed about 7-fold higher in the fluorescence than that of the original Prhl promoter. However, the leak showed no less than 25-fold high. Thus we used our improved Prhl(RL) (K1529320) in the following experiments and modelings.

2.4. HSL-dependent responses of Company E. coli with improved promoter and Customer E. coli

For construction of the C4HSL-dependent chloramphenicol resistance gene product (CmR) and 3OC12HSL production module, we designed a new part Prhl(RL)-CmR-LasI. (BBa_K1529302) (Fig. 2-1-9). In order to confirm the Company’s dependency on C4HSL, we measured the growth of Company cell in the presence and absence of C4HSL. After the induction, we added chloramphenicol into the medium and measured the optical density for about 10 hours to estimate the concentration of the cell.

Without induction of C4HSL, the cell cannot express CmR resistance gene and cannot survive in the presence of chloramphenicol. As shown in Fig. 2-1-10, when C4HSL is added to the culture, Company cell survived and increased. This result indicates that CmR was produced in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI.

To characterize the function of C4HSL-dependent 3OC12HSL production, we also performed a reporter assay by using lux reporter cell (Fig. 2-1-11). First, the expression of LasI was induced by adding C4HSL to the culture of the Company cell. Then, the supernatant of the culture was added to the culture of reporter cell. The expression of GFP in the reporter cell was measured by flow cytometer.

頑張って書いてAs Fig. 2-1-11 shows, when the supernatant of condition ??? was used, the fluorescence intensity of the reporter cell increased. Comparing the results of condition ??? and ???, reporter cell in the supernatant of the induced Company cell’s culture had ???-fold higher fluorescence intensity. This result indicates that Company cell produced 3OC12HSL in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI.

From these experiment, we confirmed that a new part Prhl(RL)-CmR-LasI synthesized CmR and 3OC12HSL as expected.

For construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL production module, we designed a new part Plux-CmR-RhlI (BBa_K1529797). In order to confirm the Customer’s dependency on 3OC12HSL, we measured the growth of Customer cell in the presence and absence of 3OC12HSL. After induction, we added chloramphenicol into the medium and measured optical density for about 10 hours to estimate the concentration of the cell.

Without induction of 3OC12HSL, the cell cannot express CmR and cannot survive in the presence of chloramphenicol. As shown in Fig. 2-1-13, when 3OC12HSL is added to the culture, Customer cell survived and grew. This result indicates that CmR was produced in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.

To characterize the function of 3OC12HSL-dependent C4HSL production, we also performed a reporter assay by using lux reporter cell (Fig. 2-1-14).

First, the expression of RhlI was induced by adding 3OC12HSL to the culture of the Customer cell. Then, the supernatant of the culture was added to the culture of reporter cell. The expression of GFP in the reporter cell was measured by flow cytometer.

As Fig. 2-1-14 shows, when the supernatant of condition ??? was used, the fluorescence intensity of the reporter cell increased. Comparing the results of condition ??? and ???, reporter cell in the supernatant of the induced Customer cell’s culture had ???-fold higher fluorescence intensity. This result indicates that Company cell produced C4HSL in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.

From these experiments, we confirmed that a new part Plux-CmR-RhlI synthesized CmR and C4HSL as expected.

2.5. Assay of symbiosis between Company and Customer by co-culture

For the accomplishment of interdependence between the Company cells and Customer cells, we mixed and co-cultured the two cells to show symbiosis of them. Company’s characteristics are C4HSL-dependent survival and 3OC12HSL production, and Customer’s characteristics are the opposite from Company’s. (If you want to know about these cells in more detail, see the above section. Each cells’ function is described.) From these characteristics, the symbiosis between the two cells can be established.

Fig. 2-1-18. The result of co-culture assay

Two types of fluorescent proteins were used to trace the growth of each cells in our symbiosis experiments. We constructed the Company cell containing GFP and Customer cell containing RFP. By measuring the fluorescence intensity of GFP with flow cytometer, the symbiosis and its condition was detected.

3. Addition of Bank

(この節のイントロのつもり)

We have demonstrated the interdependence between one pair of Company and Customer through modeling and wet-experiments. However, thousands of Company-Customer pairs exists in the actual economy. In the experimental design of such next demonstration, our modeling suggests that our educational tool of economics include an additional player, Bank.

3.1. Motivation

In the experimental design to expand the number of Company-Customer pairs in one test tube, we found that carrying capacity of a medium is shared among the pairs. In other words, the amount of cell corresponding to each pair must be less to establish the symbiosis.

    Our modeling, however, suggested that a certain amount of cells is needed to maintain the symbiosis between Company and Customer (See 2.2. Condition for the optimal growth deducted from simulation).

    We noticed that the introduction of bank may assist not only the symbiosis with small amount of cells in a test tube, but also the understanding of economics as an education tool. In real economy, a central bank will supply money to the market when money is in short supply. Therefore, we designed Bank E. coli that does the same work as central bank.

3.2. Genetic circuit design of Bank E. coli

The circuit design of Bank E. coli is shown in Fig. 2-1-16. Bank E. coli functions as a central bank to regulate the money supply in the market. Bank E. coli can change into two states by using toggle switch (Gardner, 2000), which depends on C4HSL concentration (Fig. 2-1-17).

 As shown in the figure above, when the money supply in the economy becomes lower than a certain level, Bank will supply money by expressing RhlI. This supply represents stimulation of economy. On the other hand, when money supply becomes higher than a bifurcation point, Bank E. coli will collect money from the economy by expressing AiiA. This decrease in money supply represents prevention of bubble economy. The whole mechanism this state switching is explained below in four steps (Fig. 2-1-18).

中央銀の経済用語として、distribution, collectionでいいか調べる→Distribution○、Collection△

For maintenance of symbiosis even in lack of money supply, Bank changes to the distribution state. In this step, transcription in Prhl/lac promoter is stopped by lower C4HSL concentration resolved by AiiA. Therefore, TetR will lower the expression. Instead, Ptet promoter is activated to express RhlI and LacI.

If Bank is always in the distribution state, too much money will be supplied to the economy. Thus when the amount of C4HSL is excessed which will activate Prhl/lac promoter to express AiiA. When Bank finishes this change, it will switch to the collection state.

If the amount of money in the economy become excessive, Bank will be in the collection state. In this state, AiiA in Bank will be expressed. AiiA decomposes C4HSL and 3OC12HSL in the medium to decrease the money supply in the economy.

For maintenance of symbiosis even in lack of money supply, Bank changes to the distribution state. In this step, transcription in Prhl/lac promoter is stopped by lower C4HSL concentration resolved by AiiA. Therefore, TetR will lower the expression. Instead, Ptet promoter is activated to express RhlI and LacI.

3.3 Modeling; Bank actually helps 経済の発展

まず、マネーサプライに応じて銀行がDistribution State からCollection Stateに変わるために、どのパラメタがかかわるかを調べた。

Since the Bank circuit has to change its state depending on the concentration of C4HSL, Bank has to be neatly adjusted. To clearly show which components should be concerned, we analyzed the system. The analysis shows that the most crucial point in the system is the relative intensities of Plux/lac and Ptet. Fig. 2-1-24 is the modeling result.加藤君の説明必要

If the intensities of Plux/lac and Prhl promoters are in the striped area in the figure, Bank can change its state depending on the concentration of C4HSL. The detail of this analysis is shown in the modeling page.

With these optimized components, our simulation shows that Bank behaves as expected. Fig. 2-1-25.shows that the Company and Customer cannot grow well only with themselves. However, once the Bank is included, Bank helps them to grow well as shown in Fig. 2-1-25.. The result is

During this growth, Bank changes its state from Distribution State to Collection State as shown in Fig. 2-1-26.

"These simulation shows that Bank functions as central bank in the real economy"

4.Economic Wave

We introduced our Bank E. coli project to businessmen, executives, and technicians engaged in IT companies for some professional opinions at a workshop called MUSE TALK (See Policy and Practice for more details). They pointed out that economic wave should be integrated into our system to make it more realistic (Fig. 2-1-27).

According to the advice we received, we began to consider economic waves into our project. In the real economy, there are long-term financial wave named Kitchin inventory, Kondratiev wave etc. (Wikipedia.org). These long-term financial waves deeply affect the whole economy. We were advised that ignoring these waves can be a crucial defect in the tool’s economic system.

We merged the idea of economic wave into our system as the fluctuation of the C4HSL concentration. Detailed description of our integration is shown in the modeling page.

Fig shows 波による破壊と、銀行によるフォローを示す。 波無でしうまくいっていたのが、they cannot endure the effect of the wave, especially during the recession. This results in the destruction of the whole economy. This is shown as the death of whole cells in the system as shown in Fig. 2-1-28. But with the Bank, the Company and Customer can endure the effect. This is shown in Fig. 2-1-30.

In the simulation, the amount of money represented by the concentration of C4HSL is so low that the trade between Company and Customer cannot proceed properly. In the figure, the fluctuation of the wave is the result of the economic wave (Fig. 2-1-29). Even though the amount of money still fluctuates, the average amount of money is increasing.  

The amount of money during the simulation is shown in Fig. 2-1-31.

5. Reference