In Bacillus subtilis the s B mediated general stress response provides protection against various... more In Bacillus subtilis the s B mediated general stress response provides protection against various environmental and energy related stress conditions. To better understand the general stress response, we need to explore the mechanism by which the components interact. Here, we performed experiments in B. subtilis wild type and mutant strains to test and validate a mathematical model of the dynamics of s B activity. In the mutant strain BSA115, s B transcription is inducible by the addition of IPTG and negative control of s B activity by the anti-sigma factor RsbW is absent. In contrast to our expectations of a continuous b-galactosidase activity from a ctc::lacZ fusion, we observed a transient activity in the mutant. To explain this experimental finding, we constructed mathematical models reflecting different hypotheses regarding the regulation of s B and b-galactosidase dynamics. Only the model assuming instability of either ctc::lacZ mRNA or b-galactosidase protein is able to reproduce the experiments in silico. Subsequent Northern blot experiments revealed stable high-level ctc::lacZ mRNA concentrations after the induction of the s B response. Therefore, we conclude that protein instability following s B activation is the most likely explanation for the experimental observations. Our results thus support the idea that B. subtilis increases the cytoplasmic proteolytic degradation to adapt the proteome in face of environmental challenges following activation of the general stress response. The findings also have practical implications for the analysis of stress response dynamics using lacZ reporter gene fusions, a frequently used strategy for the s B response.
Recombinant human protein disulfide isomerase (PDI) was expressed in vivo in Escherichia coli usi... more Recombinant human protein disulfide isomerase (PDI) was expressed in vivo in Escherichia coli using a non-optimised gene sequence and an optimised sequence with four 5 codons substituted by synonymous codons that take less time to translate. The optimisation resulted in a 2-fold increase of total PDI concentration and by successive optimisation with expression at low temperature in a 10-fold increase of the amount of soluble PDI in comparison with the original wild-type construct. The improvement can be due to a faster clearing of the ribosome binding site on the mRNA, elevating the translation initiation rate and resulting in higher ribosome loading and better ribosome protection of the PDI mRNA against endonucleolytic cleavage by RNase. This hypothesis was supported by a novel computer simulation model of E. coli translational ribosome traffic based upon the stochastic Gillespie algorithm. The study indicates the applicability of such models in optimisation of recombinant protein sequences.
In translation initiation the 3' end of the 16s rRNA binds to the complementary Shine Dalgarno (S... more In translation initiation the 3' end of the 16s rRNA binds to the complementary Shine Dalgarno (SD) sequence. Together with bound initiation factors translation can subsequently begin at the AUG start codon. However, there may be SD related sequences throughout the coding region of the mRNA. These secondary SD sequences can recruit ribosomes as well, in particular if they are embedded in purine rich regions . The existence of such secondary ribosome binding sites can greatly reduce the expression efficiency since ribosomes recruited to the secondary SD site hinder elongating ribosomes in their progression. If a start codon is nearby a secondary SD site even truncated protein could build up in expense of full length protein [2].
In this article we present and test a strategy to integrate, in a sequential manner, sensitivity ... more In this article we present and test a strategy to integrate, in a sequential manner, sensitivity analysis, bifurcation analysis and predictive simulations. Our strategy uses some of these methods in a coordinated way such that information, generated in one step, feeds into the definition of further analyses and helps refining the structure of the mathematical model. The aim of the
In Bacillus subtilis the s B mediated general stress response provides protection against various... more In Bacillus subtilis the s B mediated general stress response provides protection against various environmental and energy related stress conditions. To better understand the general stress response, we need to explore the mechanism by which the components interact. Here, we performed experiments in B. subtilis wild type and mutant strains to test and validate a mathematical model of the dynamics of s B activity. In the mutant strain BSA115, s B transcription is inducible by the addition of IPTG and negative control of s B activity by the anti-sigma factor RsbW is absent. In contrast to our expectations of a continuous b-galactosidase activity from a ctc::lacZ fusion, we observed a transient activity in the mutant. To explain this experimental finding, we constructed mathematical models reflecting different hypotheses regarding the regulation of s B and b-galactosidase dynamics. Only the model assuming instability of either ctc::lacZ mRNA or b-galactosidase protein is able to reproduce the experiments in silico. Subsequent Northern blot experiments revealed stable high-level ctc::lacZ mRNA concentrations after the induction of the s B response. Therefore, we conclude that protein instability following s B activation is the most likely explanation for the experimental observations. Our results thus support the idea that B. subtilis increases the cytoplasmic proteolytic degradation to adapt the proteome in face of environmental challenges following activation of the general stress response. The findings also have practical implications for the analysis of stress response dynamics using lacZ reporter gene fusions, a frequently used strategy for the s B response.
Recombinant human protein disulfide isomerase (PDI) was expressed in vivo in Escherichia coli usi... more Recombinant human protein disulfide isomerase (PDI) was expressed in vivo in Escherichia coli using a non-optimised gene sequence and an optimised sequence with four 5 codons substituted by synonymous codons that take less time to translate. The optimisation resulted in a 2-fold increase of total PDI concentration and by successive optimisation with expression at low temperature in a 10-fold increase of the amount of soluble PDI in comparison with the original wild-type construct. The improvement can be due to a faster clearing of the ribosome binding site on the mRNA, elevating the translation initiation rate and resulting in higher ribosome loading and better ribosome protection of the PDI mRNA against endonucleolytic cleavage by RNase. This hypothesis was supported by a novel computer simulation model of E. coli translational ribosome traffic based upon the stochastic Gillespie algorithm. The study indicates the applicability of such models in optimisation of recombinant protein sequences.
In translation initiation the 3' end of the 16s rRNA binds to the complementary Shine Dalgarno (S... more In translation initiation the 3' end of the 16s rRNA binds to the complementary Shine Dalgarno (SD) sequence. Together with bound initiation factors translation can subsequently begin at the AUG start codon. However, there may be SD related sequences throughout the coding region of the mRNA. These secondary SD sequences can recruit ribosomes as well, in particular if they are embedded in purine rich regions . The existence of such secondary ribosome binding sites can greatly reduce the expression efficiency since ribosomes recruited to the secondary SD site hinder elongating ribosomes in their progression. If a start codon is nearby a secondary SD site even truncated protein could build up in expense of full length protein [2].
In this article we present and test a strategy to integrate, in a sequential manner, sensitivity ... more In this article we present and test a strategy to integrate, in a sequential manner, sensitivity analysis, bifurcation analysis and predictive simulations. Our strategy uses some of these methods in a coordinated way such that information, generated in one step, feeds into the definition of further analyses and helps refining the structure of the mathematical model. The aim of the
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Papers by U. Liebal