We obtained new estrogen receptor(ER) mutants with higher EE2 affinity through computer aided protein modification, and the mutant protein called showed good binding ability to EE2 in the experiment.After comparing the simulation results for many times, we finally chose to change the site 421 of estrogen receptor to phenylalanine. The modification of this site greatly improved the binding ability and affinity of the receptor to EE2. We called the mutants ER-421F.
The kill switch could be used in all items for degradation in environment.
Targeting for sewage treatment plant and commercial use, we hope the engineered bacteria will die automatically when the EE2 is completely degraded. It struck us that negative-edge triggers in electrical circuits possess desired functions. We borrow this model and introduce negative edge triggered pulse detecting circuit shown in Fig.3(a). MazF is expressed only under the condition of both the existence as an input and subsequent elimination of EE2. The complex of EE2 and estrogen receptor (ER) binds to estrogen response element(ERE) in the promoter and initiate the expression of cⅠDN and cⅠ.
CⅠDN is a mutant of CI repressor bearing a mutation in the DNA binding surface which eliminated the binding affinity of the cⅠ mutant to operator sites but increased the affinity to non-specific DNA binding site. When the transcription starts, with a stronger RBS, many more CⅠDN molecules are present in the cell compared to CI molecules. Therefore, almost all of the monomers will form heterodimers with CⅠDN, and there will be no CI2 to activate PRM promoter. After the induction period, because of the degradation tag added with CⅠDN to ensure quick degradation of the monomeric proteins, CⅠDN molecules degrade quickly giving CI molecules a chance to form dimers and activate PRM promoter leading to the expression of mazF.
In order to have better practical value, we refer to the actual sewage plant design, set up the treatment model of estrogen sewage plant. In order to better treat estrogen in sewage, we design a water treatment cycle with computer aided signal processing.
The estrogen degradation model of sewage treatment plant takes the degradation tank as the main part, supplemented by detection equipment. If the sample is lower than the concentration requirements, it can be discharged. If it is higher than the concentration requirements, it will enter the circulating pipeline for secondary degradation.
It is mainly divided into two modules: degradation module and detection module.
The degradation module is composed of a culture tank for expanded culture of degradation engineering bacteria and a degradation tank for elimination estrogen in sewage.
The detection module is composed of sampling point, detection engineering bacteria culture tank, optical detection equipment and signal processing system.
Judge whether the sewage sample meets the standard through optical detection equipment and signal processing system.
In recent years, the application of ammonia oxidizing bacteria (AOB) to treat waste water became a big innovation and growth point in environmental protection topic.
Through document searching, we found that some kinds of AOB could co-metabolizing EE2 in existence of nitrogen sources. However, the complicated mechanism of AOB as biological chassis hinders it to effectively degrade EE2 in target sewage environment. Accordingly, we decided to construct EE2 degradation engineering bacteria by introducing amoA gene and HAO gene from Nitrosomonas europaea ATCC 19718 into E.coli BL21.
We successfully obtained the new strain NB1 and tested it’s ability to degrade ammonia as well as EE2.
