Development And Evaluation Of Super-resolution Microscopic Method For Extracellular Proteins Imaging Of Environmental Microorganism

Among various environmental treatment methods,microbial treatment has been widely used because of its thorough treatment,no secondary pollution,high efficiency,wide range of application,low cost,stability,easy operation and other advantages.Microbial extracellular proteins play an important role in the degradation and adsorption of environmental toxic and harmful pollutants.Super-resolution microscopic imaging technology breaks through the limitation of resolution of traditional optical microscope,and can realize the nanoscale imaging of living cell substructure,which is an advanced research method.However,at present,in the field of microbial extracellular protein research,the application of super-resolution microscopy has just started.There is a lack of super-resolution imaging methods for microbial extracellular protein.Faced with the complex extracellular structure of microorganisms,how to develop a method suitable for microbial extracellular protein labeling,combined with super-resolution fluorescence microscopy technology for microbial extracellular protein in situ imaging in vivo,is an important problem.In order to solve this key problem,this study designed a super-resolution imaging labeling method for extracellular protein of living microbial cells.We successfully applied this method to the super-resolution imaging research of extracellular amyloid protein Curli fiber network structure of E.coli,providing a new idea for the research of extracellular protein of living microbial cells.The main results are as follows:(1)A super-resolution microscopic imaging labeling method for extracellular proteins of living microbial cells was designed.In this method,AP-tag was selected as the fusion tag of the target protein,and biotin was bound to the AP-tag residue of the target protein by BirA enzyme expressed by microorganism.Furthermore,the monomeric streptavidin(mSA)combined with super-resolution fluorescent dye was selected as the fluorescent probe,and the biotin-avidin system was used to specifically bind the target protein,so as to realize the super-resolution fluorescence imaging labeling of microbial extracellular protein.(2)A fluorescent probe suitable for super-resolution microscopic imaging was prepared,and a STED super-resolution microscopic imaging platform was built.In this study,we used monomeric streptavidin(mSA)as fluorescent probe protein,and selected Abberior^?Star 488,NHS ester as fluorescent dye was used as fluorescent dye.Through the binding of mSA protein with fluorescent dye,mSA fluorescent probe was successfully prepared.In cooperation with Suzhou Institute of Biomedical Eengineering Technology,Chinese Academy of Sciences,the STED super-resolution microscope was built.(3)Fluorescent labeling of extracellular OmcA protein of Shewanella oneidensis MR-1 was carried out.Three different fusion sites were selected in OmcA protein structure.At the same time,in order to biotinylate the expressed OmcA-APtag fusion protein effectively,two expression patterns of BirA were constructed(AP-tag was knocked into OmcA gene of genome and the plasmid pBBR-ParcA-bir A was added to express BirA enzyme;The gene was expressed in tandem by introducing plasmid pBBR-ParcA-birA-omcA-APtag).Finally,the constructed genetically engineered S.oneidnsis MR-1 was verified by fluorescence labeling and microscopic imaging,and the further optimization strategy of fluorescence labeling method was analyzed.(4)The fluorescent labeling of E.coli extracellular Curli protein was carried out,and the super-resolution imaging of E.coli extracellular Curli protein network structure was successfully realized.We analyzed the occurrence and secretion mechanism of E.coli extracellular protein Curli.Combined with the optimization strategy proposed in research conclusion(3),we developmented the labeling strategy with CsgA protein as the labeling object.Duet(CsgA/BirA)strain was constructed based on the co-expression of CsgA-APtag and BirA.Confocal and STED super-resolution microscopy were used to carry out the extracellular Curli fluorescence labeling imaging.The results showed that the STED imaging showed the structural details of Curli network beyond confocal imaging,which proved that the fluorescence labeling method constructed in this paper was suitable for the super-resolution microscopic imaging analysis of extracellular proteins in living microbial cells.In this paper,we developed a fluorescent labeling method for microbial living cell protein,and used this method to realize the super-resolution imaging of Curli fiber of E.coli living cell,which provides a new idea for the imaging of microbial extracellular protein in situ.However,the amount of extracellular Curli protein in this study is large,which can not fully reflect the advantage of"fine"imaging in this method.It is suggested that this method should be used to study the distribution of extracellular proteins in single cell in the future.