The Study On The Restriction And Modification System Of Sorangium Cellulosum Soce157-2

According to the difference of diets,Myxobacteria is divided into two major categories which are dissolving bacteria group and dissolving bacteria cellulose group.In nowdays,Sorangium cellulousm is the only one that can decompose cellulose in the Sorangium.Sorangium cellulousm haven't improved the production of Epothilone B through the method of traditional fermentation.Therefore,improving the production of secondary metabolite with the molecular biosynthesis method is important to the Sorangium cellulousm study.Establishing the efficient genetic operation system for the Sorangium cellulousm is the basis of this studying.The genetic operation of Myxobacteria cannot be used for every kind of Sorangium cellulousm strain.We haven't found the endogenous plasmid in the Sorangium cellulousm So ce157-2 in this study.So the genetic manipulation for Sorangium cellulousm uses exogenous plasmid as vector and it is hard to use.Because So ce157-2 is not sensitive to most antibiotics,so the range of screening genetic markers is narrow.The situations above is the one thing for hard genetic manipulation,but so is the existence of restriction-modification system.The foreign DNA will be degraded without modification when it enter into bacteria cell.These are largely affect the success probability of genetic manipulation.This study will research as followings:1.This study do the physiological and biochemical reaction experiment and analyze 32 kinds of antibiotic resistance for So ce157-2.These results for the genetic operations and the exploration for restriction-modification system provide a reliable basis.Choosed the chloramphenicol as a selection marker,through the analysis of experimental results and the comprehensive consideration of predecessors' study.So ce157-2 is different from other bacterias for the slower growth,longer growth cycle and easier contamination of other bacterias.So the growth conditions of So ce157-2 has been optimized by the single factor experiment and orthogonal experiment.The following optimal experimental conditions are used: Sorangium cells were grown in the suitable medium for 72 hours in a 250 mL erlenmeyer flask with the So ce157-2 volume of 6%.Cells were grown at 32°C.The medium adjusted to pH 7.2.The strain age of the So ce157-2 is 60 h.2.Due to the high content of G+C,chloramphenicol resistance gene cannot be expressed in low content of G+C vector,so we choose the aphII as a promoter.We get the aphII promoter fragment and chloramphenicol acetyltransferase gene fragments by PCR and insert these into the plasmid pCVD442 to build the pCVD442-cat vector.We get three fragments from So157-2 genome DNA and insert it into pCVD442-cat vector respectively to get plasmid pCVD442-cat-1,pCVD442-cat-2 and pCVD442-cat-3.Genetic operation system has been established through the method of conjugal transfer,plasmid from double plasmid e.coli to So157-2 in e.coli,and the positive rate has been reached to 100%.By exploring the influence of pH in the conjugal transfer,we ascertain that the conjugal transfer efficiency is highest when the pH value is from 8.5 to 9.0.3.Maybe there are some restrictions to hinder the exogenous gene into the body of bacteria or influence the effect of the exogenous gene express normally.We find the nuclease inside and outside the bacteria by analyzing the nuclease activty in the culture medium,on the bacteria cell wall and in the bacteria.But there is no endogenous plasmid in the bacteria.We have cloned the part of gene for restriction-modification system such as methylase gene and transmethylase gene in this study.