After absorption of cesium-137 for a period of time, we need to recover these engineered bacterial to recover cesium-137, also protect these E. Coli from living in environment in case of side-effect.

Note: So far, below experiments just can be done in lab-scale and simple simulation.

We hope to use a device to aggregate E.coli to limit their movement after absorption, and this method might be one way of delete them in environment. The device which we used is biobrick BBa_K346007 by Peking Universiy in 2010, shown on the right (Fig.1).

1. Parts

In this device, Ag43 codes for protein Antigen 43. Antigen 43 (Ag43) is an abundant outer membrane protein in Escherichia coli belonging to the autotransporter family, which promotes bacterial to a cell-to-cell aggregation. Antigen 43 can be expressed on E.coli cell surface in large quantities, up to 50000 copies per cell, and is important for biofilms and infection. The expression of Agn43 is dependent on the binding of the regulator protein OxyR. When OxyR is bound to the regulatory region of Agn43, which overlaps with the promoter, it inhibits transcription. The ON phase of transcription is dependent upon Dam methylating the GATC sequences in the beginning of the Agn43 gene (which happens to overlap with the OxyR binding site). When the Dam methylates the GATC sites it inhibits the OxyR from binding, allowing transcription of Ag43.

2. Results

With this device, we could aggregate the bacteria together first. Expression of Antigen 43 is induced by IPTG for 3~4 hours. Then the bacteria with/without inducement of IPTG are left to stand for 1~3 hours to test the effect of aggregation (Fig.2).

Result verifies the effect of BBa_K346007 and picture D shows one probable kind of shape when E. Coli aggregate together in still LB medium. The aggregated colonies are small and separated, which means aggregation might incline to lead bacteria to form fluffy masses.

Next, we plan to design a model to simply simulate the kinetic state of sea. But for the limited time, the equipment of this part is not completed.

As aggregation proceed, we postulate that colonies aggregated would be separated from each other in a certain area, which means the size of colony has a limitation. According to above experiment, the colony might be seen in naked eyes and could be recovered by physical equipment.

Before our E. Coli are spread into a certain area, buoys with sonar pinger are thrown to the water, which could flush fishes and other animals, in case of our E. Coli are eaten by them. Meanwhile, the contaminant area should be separated from environment by some solutions, such as building dykes, filterable membrane, and so forth. These solutions protect the contaminants from spreading more. Then the disposing might be easier.

At the same time, special plates with antibody would be fixed in the water, from bottom to up. Then, our aggregated colonies might be combined with the plates by another antigen, which could bind the antibody of plates (we hypothesized that the colonies are not easy to be disturbed by the water current). This step utilizes combination of antibody-antigen to a second enrichment of our E. Coli.

After a period of time, operators could recover the plates which absorb E. Coli containing cesium-137 by testing the MFI (mean fluorescent intensity) of the plates. Then, our goal to recover the bacteria is possible to be achieved.

As aggregation proceed, we postulate that colonies aggregated would be separated from each other in a certain area, which means the size of colony has a limitation. According to above experiment, the colony might be seen in naked eyes and could be recovered by physical equipment.

Before our E. Coli are spread into a certain area, buoys with sonar pinger are thrown to the water, which could flush fishes and other animals, in case of our E. Coli are eaten by them. Meanwhile, the contaminant area should be separated from environment by some solutions, such as building dykes, filterable membrane, and so forth. These solutions protect the contaminants from spreading more. Then the disposing might be easier(Fig.1).

At the same time, special plates with antibody would be fixed in the water, from bottom to up. Then, our aggregated colonies might be combined with the plates by another antigen, which could bind the antibody of plates (we hypothesized that the colonies are not easy to be disturbed by the water current). This step utilizes combination of antibody-antigen to a second enrichment of our E. Coli.

After a period of time, operators could recover the plates which absorb E. Coli containing cesium-137 by testing the MFI (mean fluorescent intensity) of the plates. Then, our goal to recover the bacteria is possible to be achieved(Fig.2).

Admittedly, all the methods are models now, which are simplified from complicated environment. To be used in real environment, there would be many parameters and conditions to be searched and optimized. We hope these problems could be solved one day to make it possible to clean the cesium-137 by our "Nuclear-Leakage Rescuers".

Besides, we have designed other method to recover the E. Coli which absorb cesium-137. Generally, E. Coli and cesium are both carried away by the water with a certain direction, such as ocean current. Accordingly, we might recover our E. Coli in the downstream of the current by another device. This method avoids the former treatment with E. Coli and even if E. Coli is eaten by animals, the effect is equal to the original result without disposing of cesium-137.

First, all E. Coli could express a kind of antigen on the surface of the membrane after expression of TrkD. Both E.coli with or without cesium would be absorbed by a net-shaped equipment in the downstream, which has antibodies of the antigen fixed on the surface of the equipment. In other words, this method is like ELISA(Fig.1).

The proper device is in search now, but we have already had candidates. In 2009, UC Berkerley developed BBa_K197020 which expresses strepavidin-binding proteins on the cell surface. We decide to add this part in the downstream of trkD, so that once TrkD is expressed, the strepavidin-binding protein would be expressed at the same time (Fig.2). Then, we add strepavidins on the surface of the net-shaped equipment. As a result, it is probable to absorb our E. Coli to recover them from environment.