Favorite Tokyo Tech 2015 iGEM Team Parts

　　　　　　

Name	Type	Description	Design	Length(bp)	Experiment
BBa_K1632007	Composite	fim switch[default ON](wild type)_rbs_gfp	Riku Shinohara	1128	Work
BBa_K1632012	Composite	Pbad/araC_fimB	Riku Shinohara	1839	Work
BBa_K1632020	Composite	rbs_cmRssrA	Jun Kawamura	712	Work

2. Calculating the 4 Different Types of OD

　　　　　　

To precisely replicate the payoff matrix, we calculated the 4 different types of OD, which results from the 4 different types of damage, but the results of the modeling did not match with the results of the wet lab.

We calculated the OD after 480 minutes by using the following equations (1) ~ (10).

However, the results of the modeling did not match with the results of the wet lab.

3. Considering the Leakage of CmR

Since the results of the modeling did not match with the wet lab, assuming that there was a leakage in the promoter of the CmR, we calculated the OD considering the leakage of the promoter of the CmR, in which the results successfully matched with the wet lab.

Given the result from the wet lab that the OD of , we assumed that the promoter is activated and CmR is produced even in the absence of AHL.

Therefore, we revised the equations (1),(2) to the following (1’),(2’).

We also added the equation (11), which represents the production of CmR due to the leakage of the promotor, in the absence of AHL.

Therefore, we customized the equations (3), (4), (7), (8) to the following (3’), (4’), (7’), (8’).

The result of the calculation is shown in Fig.4-1-1-5, and in Fig.4-1-1-6. The results of the calculation were similar to the results of the wet lab, which is shown in Fig.4-1-1-3, and in Fig.4-1-1-4.

4. Two Solutions

　　　　　　

To overcome the leakage, we tried modeling two different solutions to see which one is more efficient

4.1. Solution 1: Increasing the concentration of Cm

　　　　　　

After constructing a model in which the concentration of Cm is increased, we decided that there was no need to run any experiments in the wet lab, since the obtained results were not positive.
Although up to this point, the concentration of Cm was set to 100 µg/mL, we calculated the OD, setting the concentration of Cm to 150 µg/mL.
The results are shown in Fig.4-1-2-1, and in Fig.4-1-2-2. We used the equations (1’),(2’),(3’),(4’),(5),(6),(7’),(8’),(9),(10),and (11).

OD of damage high and middle were decreased a little.

4.2. Solution 2: Tagging the CmR protein with ssrA

　　　　　　

Obtaining positive results of modeling in which we tagged the CmR protein with ssrA, successfully led to expected results in our wet lab and ultimately led to our precise replication of the payoff matrix.
As the other solution, we tagged CmR with ssrA, which is a degradation tag.
In order to reflect the effect of the ssrA tag, we customized the equations (1’), (2’) to the following (1”), (2”).

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We also customized the equation (11) to the following (11’).

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The results are shown in Fig.4-1-2-3, and in Fig.4-1-2-4.

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From the results, tagging CmR with ssrA is a more effective solution to degrade the leakage of CmR, compared to increasing the concentration of Cm.
Therefore, we tagged CmR with ssrA in our wet lab experiments. The results are shown in Fig.4-1-2-5, and in Fig.4-1-1-6.

5. Parameters

Fig.4-1-5-1. Parameters

6. Reference

　　　　　　

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