Construction Of The Defect Engineering Strain That The Spore Of Bacillus Subtilis Can Not Germinate

The spore of Bacillus subtilis is the most commonly used carrier in spore surface display technology,which can be used to achieve the purpose of spore surface display by fusing exogenous protein and spore capsid protein together.This technology can be used to display exogenous proteins,vaccines and biological agents on the surface of spore.Although spore has the characteristics of strong resistance and can deal with the extreme environment,it is also extremely sensitive to the external environment.Once the spores detect the conditions is suitable for germination,the spores germinate immediately and restore to nutritive cells.In the process of spore restoring to nutritive cells,the spore capsid protein will be degraded and detached,resulting in the material on the surface of the spore to break away from the spores and lose its activity.In order to improve the yield of spores,the fermentation medium and culture conditions of Bacillus subtilis were optimized in order to obtain high density spores.Through the single factor screening experiment,the best carbon source of Bacillus subtilis culture medium was wheat bran and the nitrogen source was corn pulp,the inorganic salts were NaCl,K₂HPO₄ and MgSO₄.By adding range single factor test to determine the optimum concentration range of each factor,then,through orthogonal test optimize the best proportioning of wheat bran,corn pulp,NaCl,K2HPO4 and MgSO₄.Because Mn²⁺has the function of promoting the formation of spores,and has no effect on the proliferation of bacteria,so we can add different concentrations of MnSO₄ to the culture medium determined by orthogonal test to determine the optimal concentration of Mn²⁺was 2.4 mmol/mL to promote the formation of spores.In this paper,the optimal spore culture medium of Bacillus subtilis 168 was determined,wheat bran was 8 g/L,corn bran was 20 g/L,NaCl was 4 g/L,K₂HPO₄ was 2 g/L,MgSO4 was 2.5 g/L,MnSO4 was 0.4 g/L.The optimum conditions for Bacillus subtilis 168 was determined by optimizing the culture conditions,the initial pH value was 7.0,the volume of liquid was 100/500 mL,the temperature was 37?and the speed of the shaking table was about 180 r/min.After optimization,the number of bacteria reached 3.9×10⁹ CFU/mL,the number of spores reached to 3.6×10⁹ CFU/mL,the sporulation rate was 92%.In this paper,by analyzing the genes related to spore germination,two genes sleB and cwlJ which is a cortical lytic enzyme during the germination of Bacillus subtilis spores should be knocked out.Fristly,the paper took Bacillus subtilis 168 genomic DNA as templates,realized the amplification of two homologous arms by using the PCR technique,named sleB1 and cwlJ1.Secondly,using kanamycin?km^r?and zeocin?zeo^r?as resistance marker genes,through overlapping PCR technique,we connected sleB1 with the kanamycin resistance?km^r?gene and cwl J1 with the zeocin resistance?zeo^r?gene,obtained sleB1-km^r and cwlJ1-zeo^r recombinant fragment,after enzyme digestion and concentration,this two recombinant gene fragments was transformed into Bacillus subtilis 168 competent cells,through positive recombinant screening,finally,we obtained spore germination defective engineering bacteria Bacillus subtilis168?sleB?cwlJ.The results showed that,by drawing the typical growth curve,the growth of the recombinant strain was about 2 hours slower than that of the original strain.By measuring the fluorescence intensity,it was found that the knockout of sleB and cwlJ gene had no effect on spore formation.The spore germination number of original strain was 5.9×10⁸ CFU/mL and the spore of recombinant strain did not germinate in LB solid medium.The spore germination number of original strain after the transformation of 4h,8h,12h and 16h was about 5.8×10⁸ CFU/mL and the spore of recombinant strains did not germinate in each time period in the maltose conversion system.By adding different substances to stimulate the spores of recombinant bacteria,it was found that the spores had almost no response to the carbon source,inorganic salts and amino acids and there was a partial response to the stimulation of lysozyme and surfactant.