Team:SYSU-China/project Cesium Absorption
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Accidentally, we found a transmembrane protein, TrkD, which is an ion channel with high affinity to cesium ion.
Protein TrkD (belongs to Kup system) is responsible for the low-affinity transport of potassium into the cell, but with high affinity to transport cesium. In contrast to Trk or Kdp, the Kup system does not strongly distinguish between the alkali cations K+, Rb+, or even Cs+. When both of Cs+ and K+ exist, the uptake of Cs+ has inhibition of that of K+. And among several proteins in Kup system, TrkD has the best effect of uptake of Cs+. Accordingly, after E. Coli approached Cs+, the expression of TrkD will help our E. Coli to absorb Cs-137.
We have designed three categories of modules - Expressional Testing Modules, Functionalizing Testing Modules and Final Functional Modules - to test the expression and function of every element and to accomplish the final function. The followings are the details of these modules.
1. Expression Testing Modules
In order to test whether the elements we obtain from E. Coli genome by PCR can express normally, we have constructed the expressional testing modules through genetic engineering methods:
TrkD(no terminator)- gfp-pUC18 (Fig.1) and
Through the appearance of the green fluorescence with the first four modules, we can know the expression of TrkD. With the Western-blot tests using the modules TrkD-pET32a, we can directly know the expression state of TrkD.
2. Functionalizing Testing Modules
For the purpose of realizing the function that TrkD can start to express under the induction of nuclear radiation, we expected to ligate RecN with TrkD. As a result, we have constructed the following modules to conduct the functionalizing tests:
RecN-TrkD-pET28a(promoter knock-out) (Fig.3)
RecN- TrkD(no terminator)-gfp-pET28a(promoter knock-out) (Fig.4)
With the first module, we could test the expression TrkD through the Cs+ absorption under the inducement of radiation. With the second module, the expression of TrkD under the radiation inducement can be reflected directly through the fluorescence of gfp.
3. Final Functional Modules
In addition to the modules constructed above, we also intend to construct the following module:
RecN-TrkD(no terminator)-ag43- pET28a(promoter knock-out)
When E. Coli consisting of this module approaches radioactive elements, the intensity of radiation is much more than distant areas. After the intensity is higher than the threshold of recNp, the high expression of TrkD and ag43 would be started. As a result, E. Coli would absorb cesium-137 around it and then integrate with each other.
We tested the expression of TrkD by examing the fluorescence intensity. Using the laser scanning confocal microscope (LSCM), we can see obvious fluorescence trkD-GFP-pUC18(Fig.1 and Fig.2). The fluorescence of trkD-GFP-pUC18, is located mostly on the membrane of E. Coli. The lac promoter, however, will express without induction of IPTG, resulting in the expression of GFP of control group, which is trkD-GFP-pUC18 that are not induced by IPTG. Consequently, we utilized the Flow Cytometer(FCM) to exam the intensity of GFP in control group and experiment group. (We are still working on that)
We planned to exam the expression of gene trkD on protein level. We ran a western blot to find out the expression of trkD in protein level.
Within the inducement of 0.1mg/mL Isopropyl β-D-1-thiogalactopyranoside（IPTG） at 18℃ for 18h, we extracted the total proteins of the E. Coli transformed with TrkD-pET32a. Western Blot results showed that the quantity of protein TrkD significantly increased after the inducement, indicating that TrkD expressed well. Additionally, sicne a basal expression caused by promoter lac in both plasmids, the control groups showed a small quantity of proteins(Fig.3).
We use TrkD-pET32a to test the function of TrkD by measuring the amount of Cs+ in the experiment group, trkD-pET32a BL21 and control group, pET32a-BL21.
We added 3ml bacteria liquid of both the experiment group and control group into 100ml LB which contains 1g CsCl for 2h. Then we induced the expression by adding IPTG (0.5mM) and shook the conical flasks for about 4h. After that, we placed them in the 37℃ incubator for 4h. Then we centrifuged the bacteria and washed them by PBS for 3 times. Then we sent the sample to the analytical center to determine the concentration of Cs.
The analysis of Cs+ absorption through TrkD indicates that the TrkD constructed in plasmid can express normally, thus the experiment group has a higher amount of absorption than that of the control group. While the reason why the control has also absorbed Cs+ is that gene trkD also exists in E. Coli's genome, so the control group can also express a certain amount of trkD to accomplish the absorption of Cs+. As a result, the absorption of the experiment group is 47.8% higher than that of the control group.
In terms of the result, we have proved that the trkD constructed in the plasmid can function normally