Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites

Alexciev, Krassimir; Uscheva, Anna; Pavlova, M. V.; Yavachev, Libert; Ivanov, Iván

doi:10.1016/0020-711x(89)90231-0

Cited by 7 publications

(2 citation statements)

References 18 publications

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“…In the construct pP 1 -(4SD)-hIFNγ a cluster of four identical SD sequences is substituted for the single SD in the previous plasmid. As shown earlier [34], the repetition of the SD sequence can have a strong suppressive effect on translation and therefore on the yield of recombinant protein. The constructs pΔP 1 -(ΔSD)-hIFNγ and pP 1 -(ΔSD)-hIFNγ are derivatives of the plasmid pP 1 -(SD)-hIFNγ in which both promoter and SD sequence (in pΔP 1 -(ΔSD)-hIFNγ) or the SD sequence (in pP 1 -(ΔSD)-hIFNγ) were deleted.…”

Section: Resultsmentioning

confidence: 77%

Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

et al. 2011

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BackgroundSegregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under non-selective conditions this segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNγ) gene placed under the control of different regulatory elements (promoter and ribosome-binding sites) were used as a model.ResultsBacterial growth and product formation kinetics of transformed E. coli LE392 cells cultivated continuously were described by a structured kinetic model proposed by Lee et al. (1985). The obtained results demonstrated that both transcription and translation efficiency strongly affected plasmid segregation. The segregation of plasmid having a deleted promoter did not exceed 5% after 190 h of cultivation. The observed high plasmid stability was not related with an increase in the plasmid copy-number. A reverse correlation between the yield of recombinant protein (as modulated by using different ribosome binding sites) and segregational plasmid stability (determined by the above model) was also observed.ConclusionsSwitching-off transcription of the hIFNγ gene has a stabilising effect on ColE1-like plasmids against segregation, which is not associated with an increase in the plasmid copy-number. The increased constitutive gene expression has a negative effect on segregational plasmid stability. A kinetic model proposed by Lee et al. (1985) was appropriate for description of E. coli cell growth and recombinant product formation in chemostat cultivations.

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Section: Resultsmentioning

confidence: 77%

Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

et al. 2011

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“…To test this, we constructed a variant of the node that kept the same relational organization but caused a drop in the levels of XylR by attenuating the protein's translation with tandem, repeated non-overlapping RBSs (Figure 3b) [28]. This modification was expected to lower translation of the downstream ORF without any effect on mRNA stability [29]. To implement this change, the Tn 7 plasmid pTn7-Pr·RBX was built as explained in the Materials and Methods section and delivered into att Tn 7 of P. putida Pu·LUX, as explained previously.…”

Section: Resultsmentioning

confidence: 99%

Increasing Signal Specificity of the TOL Network of Pseudomonas putida mt-2 by Rewiring the Connectivity of the Master Regulator XylR

Heras¹,

Fraile²

2012

PLoS Genet

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Prokaryotic transcription factors (TFs) that bind small xenobiotic molecules (e.g., TFs that drive genes that respond to environmental pollutants) often display a promiscuous effector profile for analogs of the bona fide chemical signals. XylR, the master TF for expression of the m-xylene biodegradation operons encoded in the TOL plasmid pWW0 of Pseudomonas putida, responds not only to the aromatic compound but also, albeit to a lesser extent, to many other aromatic compounds, such as 3-methylbenzylalcohol (3MBA). We have examined whether such a relaxed regulatory scenario can be reshaped into a high-capacity/high-specificity regime by changing the connectivity of this effector-sensing TF within the rest of the circuit rather than modifying XylR structure itself. To this end, the natural negative feedback loop that operates on xylR transcription was modified with a translational attenuator that brings down the response to 3MBA while maintaining the transcriptional output induced by m-xylene (as measured with a luxCDABE reporter system). XylR expression was then subject to a positive feedback loop in which the TF was transcribed from its own target promoters, each known to hold different input/output transfer functions. In the first case (xylR under the strong promoter of the upper TOL operon, Pu), the reporter system displayed an increased transcriptional capacity in the resulting network for both the optimal and the suboptimal XylR effectors. In contrast, when xylR was expressed under the weaker Ps promoter, the resulting circuit unmistakably discriminated m-xylene from 3MBA. The non-natural connectivity engineered in the network resulted both in a higher promoter activity and also in a much-increased signal-to-background ratio. These results indicate that the working regimes of given genetic circuits can be dramatically altered through simple changes in the way upstream transcription factors are self-regulated by positive or negative feedback loops.

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The Low Expression Level of Pokeweed Antiviral Protein (PAP) Gene inEscherichia coliby the InduciblelacPromoter Is Due to Inefficient Transcription and Translation and Not to the Toxicity of the PAP

Kaloyanova

Ivanov

et al. 1998

Archives of Biochemistry and Biophysics

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Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites

Cited by 7 publications

References 18 publications

Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

Increasing Signal Specificity of the TOL Network of Pseudomonas putida mt-2 by Rewiring the Connectivity of the Master Regulator XylR

The Low Expression Level of Pokeweed Antiviral Protein (PAP) Gene inEscherichia coliby the InduciblelacPromoter Is Due to Inefficient Transcription and Translation and Not to the Toxicity of the PAP

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