Modeling of the controlled expression of a harmful protein by a three-plasmid harboring system

A genetically structured mathematical model was developed and used to evaluate the influence of molecular parameters involved in the expression of a harmful recombinant protein (SPA::EcoRI). The system consists of the controlled expression of the endonuclease EcoRI cloned in the plasmid pMTC48. The control is exerted by the lambda CI repressor expressed from the plasmid pRK248cIts. The deleterious effect of the activity of the enzyme EcoRI on the host DNA is prevented by the action of the EcoRI methylase that is expressed constitutively from a third plasmid, pEcoR4. The model includes molecular mechanisms involved in the regulation of the expression of these genes and is used to determine cultural conditions that maximize the production of the recombinant protein.     

Mathematical simulation of the growth of a three plasmid harboring Escherichia coli JM109 strain and the production of the fusion protein EcoRI::SPA with a four-compartment model

In the previous papers [35, 36] the process variables of plasmid-free, one-, two- and three-plasmid harboring E. coli JM109 cells were investigated in batch and continuous cultivation as a function of the medium composition, plasmid content, dilution rate and cultivation (generation) time. In the present paper the growth of the recombinant E. coli JM109 [pEcoR4, pRK248cI, pMTC48] and the production of the fusion protein EcoRI::SPA are simulated by using a four-compartment model, consisting of the active cell components (ribosomes, mRNA, tRNA, and others) (A), the structure forming materials and chromosomal DNA (Z), the plasmid-DNA (G) and the recombinant enzyme protein (E). At the first time, all of the three plasmids: the production plasmid (Gp), the repressor plasmid (Gr) and the protection plasmid (Gs) are taken into account in the plasmid DNA-compartment of the model. The calculated and measured courses of the cell mass, the concentrations of glucose and acetate, and the products as well as the particular plasmids agree well.     

Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA.

Plasmid RK2 encodes several kil determinants whose lethal action on Escherichia coli host cells is prevented by RK2 kor genes. Here we show that the mini-RK2 plasmid, pRK248, specifies a kilB component (kilB1) in the region of the replication gene trfA. kilB1 is different from trfA and is completely encoded within the pRK248 HaeII A fragment. Transformation of E. coli cells with hybrid plasmids containing the cloned kilB1 determinant is very inefficient and results in the selection of variant kil- plasmids, many of which show genetic and physical evidence of deletions. If another pRK248 gene (korB1) is present in the cells, kilB1+ plasmids can be established at high efficiency and without any detectable changes. KorB1 is encoded by the trfB region of pRK248 because recombinant plasmids with this region are able to control kilB1 in trans. These results substantiate our earlier explanation for the structure of pRK248 and for the perplexing requirement of the trfB region in this plasmid.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Fed-batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60-L working volume airlift tower loop reactor

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60-L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony-forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl-thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria-Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity. (c) 1993 John Wiley & Sons, Inc.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Recovery of activity of the gene coding for tetracycline resistance in the plasmin pBRS188

The spontaneous recovery of activity of tet gene deleted of the promoter region was studied. Plasmid pBRS188 was used as a model for studying this problem. The plasmid has the fragment of tet gene of pBR322, from which it originates, between the sites of restriction endonucleases EcoRI and HindIII cleavage resulting in inactivation of tet promoter. E. coli cells harbouring the plasmid were shown to revert the TcR phenotype with the frequency 10(-9). The gene activation coincided with intraplasmid recombination revealed by restriction analysis. In some cases the recovery of tet gene activity coincided with the formation of multimeric plasmids.     

Identification in vivo of promoter activity in the left site of the att region of bacteriophage lambda DNA

The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.     

Isolation of the replication region of an indigenous plasmid of Rhodobacter sphaeroides

Abstract The isolation of the replication region of an indigenous plasmid of 42 kb of the phototrophic bacterium Rhodobacter sphaeroides is described. This plasmid was digested with the Bgl II restriction enzyme, ligated to the 2.7 Bgl II fragment of transposon Tn 10 , which contains the tet genes conferring tetracycline resistance, and the mixture was transformed into the Escherichia coli MC1061 strain. One of several chimeric plasmids harboring the replication region of the 42-kb plasmid obtained by this process was named pUA33 and further characterized. Plasmid pUA33 is approx. 8.3 kb. A partial restriction map has been constructed. Plasmid pUA33 is stable in E. coli cells growing under non-selective conditions and is non-self-transmissible. All these data suggest that the pUA33 plasmid may be a very useful tool for gene cloning in R. spheroides .     

Stability fluctuations of plasmid-bearing cells: immobilization effects

The maintenance of the plasmid vectors pTG201 and pTG206 (which both carry the Pseudomonas putida xylE gene) and pB lambda H3 in Escherichia coli hosts was studied in free and immobilized continuous cultures. pTG201, containing the strong lambda PR promoter, was more quickly lost than plasmid pTG206, containing the tetracycline resistance gene promoter. The instability of pTG201 seems to be related to high expression of the cloned xylE genet. Fluctuations in the proportion of pTG201-containing cells were observed in the free system, suggesting the appearance of adaptive descendants (with and without plasmid) from the initial strains. The loss of plasmid vectors from E. coli cells and the fluctuations in the proportion of plasmid-containing cells could be prevented by immobilizing plasmid-containing bacteria in carrageenan gel beads.     

Stability of pBR322-derived plasmids

The stability of pBR322-derived plasmids was studied during growth of their Escherichia coli host in the absence of antibiotics. Plasmid pBR322, as well as its delta rom and delta bla derivatives, were lost from their host within 60 generations, but a number of delta tet derivatives were quite stable under the same conditions. An evaluation of the data indicated that primary plasmid loss due to random partitioning corresponds to the generation of a plasmid-free cell about every 10(4) divisions (probability P0; = "intrinsic" instability). Secondary loss of plasmid-carrying cells resulted from a growth advantage of the plasmid-free cells when bacteria die, perhaps due to unrepaired lethal damage in the DNA, under conditions of stationary incubation (= "apparent" instability). This cell death also occurred in the absence of plasmids but was accelerated by the presence of extra plasmid DNA in the cell and further accelerated by a functional tet gene. This was the reason for the differential apparent stabilities of delta bla and delta tet plasmids. There was no indication that an accumulation of plasmid multimers contributed to the plasmid instability, as has been suggested in the literature. The value of P0 = 10(-4) is 14 orders of magnitude greater than expected under the assumption of a random (Poisson) distribution of plasmid copy numbers in a population of cells.     

Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter.

Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.     

Expression of incompatibility by derivatives of the broad host-range inc P-1 plasmid RK2.

Summary A segment of DNA encoding incompatibility on the inc P-1 plasmid pRK248 was identified by the analysis of deletions generated in vitro, and then cloned into several unrelated and mutually compatible plasmids. These derivatives were tested for expression of P-1 incompatibility. It was demonstrated by transformation experiments that P-1 plasmids were efficiently eliminated from an E. coli host following introduction of any one of the derivatives. However, all the derivatives were compatible with each other. The cloned segment of pRK248 DNA is itself capable of autonomous replication, without being cloned into any plasmid, if plasmid-specified gene products are provided in trans. This satellite plasmid is eliminated from the cell by the inc P-1 plasmid pRK286. The results argue against a partitioning mechanism as the basis for P-1 incompatibility but are consistent with incompatibility being the consequence of negative regulation of copy number. For the inc P-1 system, susceptibility of the plasmid to elimination, but not its ability to eliminate, requires that the P-1 replication system is active.     

Survival of and plasmid stability in Pseudomonas and Klebsiella spp. introduced into agricultural drainage water

Cell survival and plasmid stability in Pseudomonas fluorescens R2f and Pseudomonas putida CYM 318 containing respectively, plasmid RP4 and pRK2501, and Klebsiella aerogenes NCTC 418 harboring plasmid pBR322 were studied in sterile and nonsterile agricultural drainage water under both aerobic and anaerobic conditions and in the absence and presence of added nutrients. Both Pseudomonas strains survived well in sterile drainage water incubated aerobically, with or without added nutrients. However, Klebsiella aerogenes NCTC 418 (pBR322) only survived in the presence of added nutrients. Pseudomonas fluorescens R2f (RP4) and K. aerogenes NCTC 418 (pBR322) did not survive under anerobic conditions without added nutrients, but showed good survival in the presence of nutrients. Survival of all three strains was negatively affected in nonsterile agricultural drainage water when compared with survival in sterile water. Maintenance of the three plasmids was host, plasmid, and environment dependent. Plasmid pBR322 was not stably maintained in K. aerogenes NCTC 418 under all conditions used in the study, and pRK2501 was readily lost from P. putida CYM 318. Maintenance of RP4 by P. fluorescens R2f was markedly influenced by added nutrients, which caused a loss of the plasmid from cells. The results of the present study demonstrate the influence of nutrients, O2, and native microorganisms on the survival of introduced bacterial strains and plasmid stability in agricultural drainage water.     

Continuous production of restriction endonucleases: continuous two-stage cultivation with E. coli JM103; continuous cell disintegration and purification by affinity chromatography

The optimization of the production of recombinant DNA-derived proteins in Escherichia coli was investigated. We chose restriction endonucleases EcoRI and EcoRV from E. coli as model proteins, despite the observation that overproduction can result in a toxic effect to the cells. The enzymes were expressed as fusion proteins consisting of protein A from Staphylococcus aureus and the desired enzyme in order to facilitate purification. The expression of the fusion protein was induced by a temperature shift using the p_R promoter of phage lambda regulated by the repressor plasmid pRK248cI. Data from batch fermentations provided the basis for planning a continuous two-stage fermentation. The EcoRI enzyme activity was investigated as a function of the induction time after cell disintegration and allowed an estimation of yield of the continuous culture. Plasmid instability, which was only observed under continuous conditions, could be prevented by adding tetracycline (resistance of the repressor plasmid) to the medium. We established a continuous cell disintegration system and purified the fusion protein semicontinuously by affinity chromatography. The biological activity of the fusion protein was the same as the native endonuclease so there was no need for cleavage of the fusion protein and the product could be used without further processing.Correspondence to: K. Schügerl     

Structural-functional organization of the incompatibility group P plasmid pBS221

Plasmid pBS221 was physically mapped for restriction endonucleases EcoRI, BamHI, BglII, HindIII. The regions essential for the plasmid existence and participating in replication (oriV trfA*) and mobilization (mob) were cloned. The tet determinant and oriV trfA* regions were localized on the physical map of the plasmid. A DNA sequence homologous to genes of Tn501 mer operon was detected in this plasmid. The studies on homology of plasmids RP4 (IncP alpha), R751 (IncP beta) and pBS221 plasmid suggest that the latter belongs to the IncP beta subgroup.