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未定

How modeling led to tagging the CmR protein with ssrA.

　　

contents

1. Introduction

2．Calculating the 4 Different Types of OD

3. Considering the Leakage of CmR

4. Two Solutions

4.1. Solution1: Increasing the concentration of Cm

4.2. Solution2: Tagging the CmR protein with ssrA

5. Parameters

6. Reference

　　

1. Introduction

　　　　　　

From our modeling which objective was to precisely replicate the payoff matrix, we found that there was an unexpected leakage in the promoter, since the results of the modeling we had done beforehand, did not match with the results of the wet lab. After modeling 2 different solutions to overcome the leakage, we found the most efficient solution of tagging the CmR protein with ssrA, which successfully led to expected results in our wet lab and ultimately led to our precise replication of the payoff matrix.

We could not obtain positive results in our modeling by increasing the concentration of Cm, which was the other solution, so we confirmed that tagging the CmR gene with ssrA is a better solution.

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

Fig.4-2-2-1. The OD 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”). 画像 We also customized the equation (11) to the following (11’). 画像 The results are shown in Fig.4-1-2-3, and in Fig.4-1-2-4. 画像 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 　　　　　　 ここにコピペ。