Construction And Application Of Plasmid PSPU237 Which Is Used To Express Gene In Saccharopolyspora Erythraea

To establish a gene transfer system in Saccharopolyspora erythraea.a producer of erythromycin,several methods including PEG-mediated protoplasts transformation,conjugal transfer as well as electroporation were investigated.Restriction barriers inherent in some streptomyces could be circumvented by conjugation or electroporation. From some elements which affect the efficiency of conjugation,included the temperature and time of pre-growth,solid medium and plasmid for transferring,will reveal the possibility of transferring the plasmid to saccharopolyspora erythraea from E.coli,but no transformant is obtained.At the same time,we explored the possibility of transferring the plasmid to saccharopolyspora erythraea via the electroporation.We also consider the three mycelium conditions included mycelium,single spore and protoplast which are used to be receptors,while we investigate the transforming effect of different plasmids(pCS5,pIJ702, pKC1139),under different voltage(700v,800v,900v, 1000v, 1100v, 1200v),and different buffer(PGS, EPB),but unfortunately we did not get the transformant.The transformation of the protoplast which PEG-mediated,currently,genetic transferring system that is used the most widely in Streptomyces.We tried to apply plasmids to transfer these protoplasts.This experiment think of the key factors included the species of plasmids,the activity of DNase,the covering time of antibiotics, restriction system that affect the transferring of Streptomyces' protoplasts to see the transforming situation of Streptomyces' protoplasts,but no transformation is obtained.It probly because of the low regeneration frequence of Saccharopolyspora erythraea' protoplast.Parameters affecting protoplast formation and regeneration of Saccharopolyspora erythraea were then studied in detail.Medium CP appeared to be more suitable than R₂,GMP,SGGP,SM,YEME for protoplast formation.The mycelia of Saccharopolyspora erythraea training adopts second to train,namely 28°C trains 72h with CP culture,5% transfers to medium CP which adds 0.5% glycine in the culture, 28°C continues training 24h.The washed mycelia were incubated in buffer P contain 2mg/ml lysozyme at 37°C for 1h.Under this condition,the preparation amount of the plotoplasts was 8×10⁸/ml.Through to the investigation of the density of sugar in washing buffer,the density of Ca²⁺,Mg²⁺ and sugar,the nutritionation...