Development And Characterization Of In Vivo And In Vitro Surface Display Systems Of Bacillus Using An Autolysin Anchor

The spore-forming Bacillus thuringiensis is an agriculturally important bacterial species that have been widely exploited for a variety of applications. It also has been generally regarded as an attractive host for developing a cell surface display system particularly a multifunctional system that would extend its agricultural or biotechnological applications, in view of its rigid cell-wall structure without outer membrane envelopes, and its unique ability to synthesize a large amount of parasporal crystal proteins. In this dissertation, a widely distributed cell wall-bound protein in various Bacillus strains was used as the anchroing motif, to consecutively immobilize the green fluorescence protein (GFP) and a mutated bacterial laccase (WlacD) onto B. thuringiensis vegetative cells and spores in vivo, and onto vegetative cells and spores of B. thuringiensis and B. subtilis, as well as the cells of several other Gram-positive and Gram-negative bacteria through an external immobilization in vitro.Mbg is a previously characterized autolysin of B. thurigiensis wild-type strain YBT-1520, which has been identified the capacity of cell-wall binding. The protein structure prediction analysis showed that Mbg is a N-acetylglucosaminidase, which can be distinguished structurally as the C terminal domain with the predicted catalytic activity, and the N terminal domain with cell-wall binding capacity by using its two LysM motifs. Based on this analyses, RT-qPCR was initially performed to determine the transcriptional activity of Mbg encoding gene, mbg, in a successive culture period of the host strain for 7 days, revealed that this gene reached a maximum activity at the growth phase of 80% spores released.In this study, the fragment Pcry3Aa-mbgn-gfp was inserted into pHT304, resulting in recombinant plasmid pMB164. Transformed B. thuringiensis BMB171 strains harboring plasmid pMB164 was designated as MB 164. In MB 164, Mbg can direct the consecutive immobilization of GFP onto B. thuringiensis vegetative cells and spores using the N-terminal domain (Mbgn). Immobilization in vivo of Mbgn-GFP fusion proteins conferred the active GFP on the surface of vegetative cells and spores, which were confirmed by fluorescence intensity or enzymatic activity measurement, immuofluorescence microscopy and flow cytometry assays. In order to validate and analyse further, construct the expression purificated carrier of fusion protein pMB313, fusion protein Mbgn-GFP connect to the pET22b(+) which is digested by the same enzyme. We do in vitro immobilization on cells and spores using purificated fusion protein Mbgn-GFP, the fluorescene intensity of Bt vegetation cells can reach 180, and the Bt spores can reach to 140 the same with Bs vegetation and spores. This in vitro immobilization still can be realized in other kind of Gram-positive Bacteria, proved the catholicity of the immobilization function.Then, using laccases protein WlacD as function protein replace the report protein GFP to construct Mbgn-WlacD surface display system. Amplify the mutation laccases gene WlacD, then connect to the pMB164 which is digested by the same enzyme. The recombinant plasmid was named pMB316, transform into Bt getting the recombinant bacterial MB316. To assay the laccases activity of recombinant bacterial cells, MB316 vegetation can reach 10.56 U/ml outclass the comparsion strains BMB171(1.148 U/ml),and the spores (4.648 U/ml)also have a certain activity. Later we use immunofluorescence microscope and flow cytometer assay(the fluorescence efficiency was 33.43%) and the consequence show that in vivo expression of Mbgn-WlacD fusion protein was not only anchored on the vegetative cells'surface on a high level, but also WlacD display on the cell surface as its activity form.We also use purificated fusion protein to do the immobilization in vitro which is on the cells and spores. Construct the purificated carrier of Mbgn-WlacD fusion protein and amplificate the fusion gene of mbgn-wlacD using pMB316 as template. After using EcoRⅠ/HindⅢdigest the amplicated production, collected to pTrcHis C, then the production is called pMB317. After using Mbgn-WlacD puricated protein incubate.The enzyme activity of Bt vegetation and spores,Bs vegetation and spores were2.74 U/ml,1.86U/ml,2.22 U/ml,1.72 U/ml, respectively. But the unincubation vegetation and spores of BT were only 1.15U/ml,0.744U/ml, respectively. So we comfirmed that Mbgn as a carrier protein was widely used.