Differences Between The Scaling-up Of Two Recombinant Escherichia Coli And Optimization Of Temperature Induction

The recombinant genetically engineered bacteria had significance and valuble,it must be stable and has genetic character.Therefore,recombinant genetically engineered bacteria required to be stable in use without mutation or attenuation or disappearance of certain characteristics.In this study,the stability of strain 1 and strain 2 were investigated in the passage process,fermentation culture process and freezing preservation process,and the results were as follows:1.The recombinant genetically engineered bacteria?strain 1 and strain 2?showed good stability during the passage in LB liquid medium.The passage stability of strain 1 is100%for 50 generations and 99%for 60 generations.The passage stability of strain 2 was100%for 40 generations and 99%for 60 generations.2.Recombinant genetically engineered bacteria?strains 1 and strain 2?inevitably lost their plasmids during fermentation.At batch phase,there was almost no plasmid loss in strain 1 and strain 2 cells.In the feeding phase,both strain 1 and strain 2 showed plasmid loss as the feeding time progressed.The proportion of strain 1 without loss of plasmid cells in the later feeding phase was 91%,and that of strain 2 was 96%.After 42?temperature induction,both strains plasmid loss were evident,finally the proportion of strain 1 without loss of plasmid cells was 84%,and that of strain 2 was 80%,compared with the strain 1,strain 2 plasmid lost more.3.The recombinant genetically engineered bacteria was preserved in glycerol cryopreservation,preserved at different conditions,the results were significantly different:Preserved at 4?,the number of living cells of both strains was stable within 30 days,but that decreased significantly after 30 days.Preserved at-20?,the number of living cells of both strains were decreased slowly within 12 months,but that decreased significantly after12 months.Preserved at-80?or liquid nitrogen?-196??,the number of living cells of both strains were decreased slowest,strains preservation stability were better,the stability was best preserved at liquid nitrogen especially.In addition,during the preservation of strains,repeated freezing and melting would seriously reduced the survival rate of recombinant escherichia coli,because every freezing,the water of cell formed ice crystals,and ice crystals damaged cells.Therefore,in order to make a high survival rate,the recombinant escherichia coli should not be repeated freezing and melting.During the fermentation amplification of recombinant genetically engineered bacteria?strain 1 and strain 2?,the differences between the two strains were as follows:1.During the common medium fermentation in the shake flask,the concentration of human-like collagen in strain 1 was 0.23 g/L,and the concentration of human-like collagen in strain 2 was 13.04%higher than that in strain 1.During the optimized medium fermentation in the shake flask,the concentration of human-like collagen in strain 1 was0.21 g/L,and the concentration of human-like collagen in strain 2 was 33.33%higher than that in strain 1.From the results of fermentation in the shake flask,strain 2 was more advantage than strain 1.2.In the 30 L fermentor,the growth of cells and the expression of human-like collagen were significantly different at different specific growth rates:when the specific growth rate of strain 1 was 0.20 h^-11 and 0.25 h^-1,the concentration of cell and human-like collagen was well?83.7 g/L,12.8 g/L and 83.8 g/L,12.5 g/L,respectively?,when the specific growth rate of strain 2 was 0.15 h^-1,the concentration of cell and human-like collagen was maximum?84.6 g/L,13.7 g/L?.3.In the process of amplification from 30 L fermentor to 125 L fermentor,the growth of cells and expression of human-like collagen of both strains were obvious decreased,the cell concentration of strain 1 decreased by 2.86%and the expression of human-like collagen decreased by 36.56%.The cell concentration of strain 2 decreased by 14.64%and the expression of human-like collagen decreased by 32.04%.One of the reasons may be that the oxygen transfer capacity of 125 L fermentor was lower than that of 30 L fermentor.4.Since the fermentation of strain 2 was more stable,strain 2 was selected for two-step temperature-upshift induction optimization,and the results were as follows:for the first time in 42?induction time 3 h was best,At the end of fermentation,the concentration of human-like collagen was 12.58g/L,and that of the comparison group was 10.36 g/L,which was 21.43%higher than that of the comparison group.The two-step temperature-upshift induction caused certain pressure on the growth of cells,and the final cell concentration was only 87.83%of that of the comparison group.