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1.
The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.  相似文献   

2.
Recombinant pTG201 plasmid coming from pBR322 plasmid, has been incorporated into Escherichia coli K12 to determine the influence of several culture conditions on the variation of the copy number. Continuous and batch cultures on LB medium without antibiotic selection and different oxygen tensions (21% and 100%) have been tested. The expression of pTGH201 encoded genes and the kinetics of plasmid loss differ significantly from the behaviour of pBR322 plasmid.  相似文献   

3.
Summary Cells of Escherichia coli K12, carrying the recombinant plasmid pTG201, were immobilized in -carrageenan gel in order to improve the following plasmid parameters: (i) maintenance of a high level of plasmid copy number, (ii) good plasmid stability and (iii) good expression of plasmid encoded gene. The experiments were carried out on LB medium without antibiotic selection in continuous and batch cultures supplied with air or pure oxygen. Parallel experiments with free cells were also performed. In all the cases immobilized cells presented better plasmid stability parameters than free cells. Best results were obtained with immobilized cells supplied with pure oxygen. In this case, an average plasmid copy number of 60 and a value of plasmid-carrying cells close to 100% were maintained with little change during more than 200 generations. In addition, an optical microscopy analysis is proposed to allow the quantitation of cell growth in gel beads.  相似文献   

4.
通过对产普鲁兰酶的重组大肠杆菌E.coli BL21(DE3)/p ET28a-s-pul菌株在发酵过程中质粒稳定性和普鲁兰酶生成量的考察,发现不同宿主对质粒稳定性及酶活性有重要影响。本文利用E.coli BL21(DE3)p Lys S菌株为宿主,构建重组菌E.coli BL21(DE3)p Lys S/p ET28a-s-pul,通过控制外源蛋白的本底表达,提高了重组菌株的质粒稳定性。优化发酵培养基和发酵条件以后,重组菌产普鲁兰酶能力由480 U/m L提高至627 U/m L,增幅为30.6%。研究结果认为,严格控制外源蛋白的本底表达,是改善重组菌稳定性的重要方法之一。  相似文献   

5.
6.
Escherichia coli BZ 18 harboring the plasmid pTG 201 and immobilized in carrageenan gel beads in continuous culture without selection pressure, provides a better stability of the plasmid than free cells, with an approximately equal production of biomass.  相似文献   

7.
A recombinant plasmid was constructed by ligating EcoRI digests of the plasmid cloning vector pBR325 and pZMO2, one of the natural plasmids of Zymomonas mobilis ATCC 10988. This vector, named pDS212 (total size 7.9 kb), which was able to transform Escherichia coli efficiently, was also transferred to Z. mobilis hosts by mobilization during conjugation using the helper plasmid pRK2013. pDS212 was inherited stably in both E. coli and Z. mobilis hosts and could be recovered intact from them. Markers of pBR325 and pRK2013 were also transferred in Z. mobilis but at very low frequencies. Neither pBR325 nor pRK2013 could be recovered intact from the Z. mobilis hosts. It is proposed that expression and stability of pDS212 in Z. mobilis is due to the origin of replication of pZMO2 that it carries, and that it may be used for developing a gene transfer system in Z. mobilis.  相似文献   

8.
Y Tierny  C G Hounsa  J P Hornez 《Microbios》1999,97(386):39-53
The genes encoding pectin methylesterase (pme) and pectate lyase (pel) from Bacteroides thetaiotaomicron were previously cloned in Escherichia coli. In the absence of selective pressure the recombinant vectors harbouring a functional pel gene were rapidly lost. This instability was due to a toxic effect of the pel gene product when overproduced and was closely related (1) to a decrease of the growth rate, and (2) to the impossibility of transforming different strains of E. coli with the recombinant plasmids harbouring a functional pel gene. When the expression level of the pel gene was reduced and the tet gene partially deleted, the stability was greatly improved. The export of pectate lyase in the extracellular medium was significantly enhanced in the presence of glycine with a positive effect on plasmid stability for low concentrations. Furthermore, using a factorial design at two levels, the effects of tetracycline, ampicillin, glucose and magnesium on pBT4 stability were quantified.  相似文献   

9.
The effect of dissolved oxygen shock on the stability of recombinant Escherichia coli cells containing plasmid pKN401 was investigated. The recombinant cells were stable in control batch experiments in media with and without ampicillin. However, these recombinant cells were highly unstable under conditions where a dissolved oxygen shock was induced. The results have implications for design of aerated reactors for recombinant cells.  相似文献   

10.
The effect of higher cell densities on the expression and segregational stability of a recombinant E. coli- B. subtilisshuttle plasmid coding for carboxymethylcellulase (CMCase) activity, was studied in E. coli DH5. Of the various feeding policies adopted for maximal expression and stability, exponential feeding resulted in the highest biomass of 15g dry cell weight (DCW) l–1 and plasmid stability of 45%. A CMCase activity of 11400 Uml–1 was achieved as compared to 230 Uml–1 during batch cultivation. In the case of other feeding strategies viz., constant feeding, linear feeding or intermittent feeding, the plasmid stability varied between 20% to 60%. Biomass achieved ranged from 5.0 g DCW l–1 to 9.0 g DCW l–1 and enzyme activities were between 2550 Uml–1 and 6000 Uml–1.  相似文献   

11.
We found that lambda plasmid replication, as measured by the increase in plasmid content per bacterial mass, proceeds for hours in an amino acid-starved, relaxed mutant of Escherichia coli K-12, whereas is inhibited in its wild-type stringent partner. Replication of lambda plasmid in amino acid-starved, relaxed cells reveals absolute lambda O dependence and is not inhibited by chloramphenicol at 200 micrograms/ml. The replication also occurs in wild-type cells treated with chloramphenicol. We conclude that lambda plasmid replication is under stringent control, probably as a result of the action of ppGpp, the signal for the stringent response, on RNA polymerase.  相似文献   

12.
U E Cheah  W A Weigand  B C Stark 《Plasmid》1987,18(2):127-134
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13.
14.
The influence of biotic and abiotic factors on plasmid transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in transfer frequency was investigated. The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1. As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10. The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1. However, the transconjugants were detected even with less than 1 mg of C liter-1. Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001). Neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. We can conclude that plasmid transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.  相似文献   

15.
The influence of biotic and abiotic factors on plasmid transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in transfer frequency was investigated. The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1. As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10. The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1. However, the transconjugants were detected even with less than 1 mg of C liter-1. Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001). Neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. We can conclude that plasmid transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.  相似文献   

16.
Recombinant E. coli strains, obtained by gene engineering techniques and capable of producing human alpha-interferon and HIV proteins, were studied. The cultures under study were completely eliminated from the body of experimental animals (mice) in 48 hours, and generalization of the infectious process took place. The study revealed that these recombinant strains had low virulence and were weakly adhesive, nontoxigenic and weakly toxic. Thus, the recombinant strains under study could be classified with class 3-4 of danger according to the "Classification of Strains of Industrial Microorganisms by the Degree of Their Danger".  相似文献   

17.
The conjugative transfer of plasmid RP4 between two strains of Escherichia coli in a sterile marine sediment was enhanced by the presence of glycine betaine (frequency increased 20 to 40 times). The conjugation was also facilitated by the osmoprotection of donor cells. Glycine betaine is a universal osmolyte and has been found in marine sediments at high concentrations. So this phenomenon could have epidemiological and sanitary importance by increasing the possibility of dissemination of some plasmids present in enterobacteria in natural marine deposits.  相似文献   

18.
Tang  Xiao-Ling  Hu  Wen-Ye  Wang  Zhi-Chao  Zheng  Ren-Chao  Zheng  Yu-Guo 《Biotechnology letters》2021,43(7):1265-1276
Objective

To solve the bottleneck of plasmid instability during microbial fermentation of l-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase.

Results

The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for l-DOPA biosynthesis.

Conclusions

The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for l-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.

  相似文献   

19.
M Khosravi  W Ryan  D A Webster  B C Stark 《Plasmid》1990,23(2):138-143
We have previously found an inverse relationship between certain cell growth parameters and plasmid size for a series of recombinant Escherichia coli strains containing pUC8 or one of a series of pUC8 recombinant derivatives. To extend these results we investigated whether there was a similar variation among our strains in oxygen requirement, which might be related to the differences in growth. During logarithmic growth in shake flasks, oxygen uptake by E. coli strain JM103 containing an 8.7-kb pUC8 derivative (pBS5) was 2.5 times that of JM103 harboring pUC8 (2.7 kb) and 7.5 times that of plasmid-free JM103. Supplementing the medium with acetate eliminated both the growth disadvantage of and the increased oxygen uptake by the strain harboring pBS5 compared with that containing pUC8. In all cases oxygen consumption decreased drastically as cells began and then continued into stationary phase, and no significant difference was seen among the three strains at these times. When the three strains were grown in a fermentor with continuous monitoring of oxygen levels, plasmid-free JM103 outgrew JM103 containing pUC8 or pBS5 at three levels of aeration. The latter two strains grew identically when aeration was high; their growth curves diverged, however, when aeration was low. In the fermentor experiments the point at which the growth of the three strains diverged was coincident with the point of oxygen depletion in the cultures.  相似文献   

20.
Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains.  相似文献   

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