Stability Of Bacillus Thuringiensis Replication Origin Ori60 Drived Recombinant Plasmids With Various Size

Our lab have completed the genome sequence of Bacillus thuringiensis YBT-1520 and plasmid copy numbers (PCNs) of 11 native plasmids, and discovered PCN was negatively correlated to the molecular mass of plasmids, plasmid DNA amount was greater than Chromosome DNA amount. Monitoring the PCN of smallest plasmid pBMB2062 in different hosts, the result indicated that the PCN of the same plasmid varies in different hosts. Due to the 11 native replication origin were different, what would happen about the PCNs of plasmids with the same replicon? This paper regarded replication origin ori60 of pBMB95 in YBT-1520 as the reseach object, and the results were summarized as following:1. Construction of various sizes recombinant plasmid with Bt replication origin ori60We selected various-sizes plasmids from BAC library in our lab, and substituted pEMB0557 for E coli BAC vector. Finally various-size recombinant plasmids were obtained:pBMB10 (10kb), pBMB19 (19kb), pBMB36 (36kb), pBMB45 (45kb), pBMB55 (55kb), pBMB75 (75kb), pBMB85 (85kb). And then they were electro-transferred into acrystalliferous mutant strain BMB171. The recombinant strains were named respectively:BMB010, BMB019, BMB036, BMB045, BMB055, BMB075, BMB085.2. Plasmid stability detection in different temprature and different mediumThree different recombinant strains (BMB019, BMB055, BMB085) were cultured in three different temperature (20℃,28℃,37℃) and determined by plasmid segregational stability detection. As BMB019 for example, Cultured in the condition of 28℃and 20℃, plasmid stability was descended to 10%. But plasmid stability obviously descended to 90% when cultured in the condition of 37℃.Choosing 4 species of common medium for culturing Bacillus, as followed:LB medium, T3 medium, glucose-yeast extract medium and NYS/CAA medium, and then determined by plasmid segregational stability detection.As BMB019 for example, plasmid stability was descended to 10% after 100 generations in LB medium; in T3 medium, plasmid stability was descended to 80%; a glucose-yeast extract medium and NYS/CAA medium were the worst,95%.Analysis of the composition of the medium shows category of mineral salt ions and organic nutrients content had certain influence in the plasmid segregational stability and that mineral salt ions had negative effect on the plasmid stability. Organic nutrients content had positive effect on plasmid stability.3. PCN and segregational stability determination of various-size plasmidsThrough PCN determination by real time-qPCR, we discovered that the PCN negatively correlated to the molecular mass of plasmids. This negative correlation appears to be linear. This study about the effect of the size on PCN ofθ-type plasmids was firstly reported.We discovered that the plasmid stability were negatively correlated to the molecular mass of plasmids. This negative correlation appeared to be nonlinear but inverted S-type. This study about the effect of the size on plasmid stability of 0-type plasmids was firstly reported.4. Location of the plasmid pBMB28 separation stability factorWith the chromosome walking strategy, four clones covering the full length of plasmid pBMB28 were screened from the BAC library. We detected the four plasmids segregational stability, and then found that two plasmids (pBMB625, pBMB721) maintenance were 100%. We regarded pBMB625 as our object, and located stability factors at 9 kb DNA fragment by lopping, By DNA sequence blast analysis and Amino acid sequence alignment analysis, we speculated that it might be a new plasmid distribution system.These findings described above additionally helped us understand PCN and segregational stability.