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Non-Invasive Analysis of Recombinant mRNA Stability in Escherichia coli by a Combination of Transcriptional Inducer Wash-Out and qRT-PCR.

Kucharova V, Strand TA, Almaas E, Naas AE, Brautaset T, Valla S - PLoS ONE (2013)

Bottom Line: The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case.We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system.The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.

ABSTRACT
mRNA stability is one among many parameters that can potentially affect the level of recombinant gene expression in bacteria. Blocking of the entire prokaryotic transcription machinery by addition of rifampicin is commonly used in protocols for analysis of mRNA stability. Here we show that such treatment can be effectively replaced by a simple, non-invasive method based on removal of the relevant transcriptional inducers and that the mRNA decay can then be followed by qRT-PCR. To establish the methodology we first used the m-toluate-inducible XylS/Pm expression cassette as a model system and analyzed several examples of DNA modifications causing gene expression stimulation in Escherichia coli. The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case. To support the experimental data a simple mathematical fitting model was developed to calculate relative decay rates. We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system. The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems. Moreover, as expression systems based on diffusible inducers are almost universally available, the concept can be most likely used to measure mRNA decay for any gene in any cell type that is heavily used in molecular biology research.

No MeSH data available.


Related in: MedlinePlus

Kinetics of gm-csf and ompA-gm-csf transcript decay, expressed from Pm.The larger plot shows decay kinetics with both gm-csf and ompA-gm-csf transcript levels at time point zero arbitrarily set to one. The smaller plot shown in the upper right corner represents all transcript data relative to the value of gm-csf at time point zero (arbitrarily set to one). Solid lines represent the best fit to the data calculated according to Eqn 4 (Material and Methods). Error bars show the deviation between two biological recurrences. RQ: relative quantification, AU: arbitrary units.
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pone-0066429-g004: Kinetics of gm-csf and ompA-gm-csf transcript decay, expressed from Pm.The larger plot shows decay kinetics with both gm-csf and ompA-gm-csf transcript levels at time point zero arbitrarily set to one. The smaller plot shown in the upper right corner represents all transcript data relative to the value of gm-csf at time point zero (arbitrarily set to one). Solid lines represent the best fit to the data calculated according to Eqn 4 (Material and Methods). Error bars show the deviation between two biological recurrences. RQ: relative quantification, AU: arbitrary units.

Mentions: In a previous study [15], we showed that expression of the gm-csf gene, encoding human granulocyte-macrophage colony-stimulating factor (GM-CSF), is strongly stimulated at the transcript and protein levels, by fusing the ompA translocation signal sequence in frame with the 5′ end of the gm-csf coding sequence. This therefore represented a different test case for investigation of mRNA stability and its potential role in recombinant protein production. The decay of gm-csf transcript was monitored in parallel for DH5α (pGM29) and DH5α (pGM29ompA) strains. The results showed that the addition of ompA 5′ signal sequence leads to a significant increase in the gm-csf mRNA stability (Figure 4), estimated to be a 4-fold decrease in the relative decay rate; 0.31 for gm-csf (95% CI = 0.30–0.33) and 0.08 for ompA-gm-csf (95% CI: 0.08–0.09).


Non-Invasive Analysis of Recombinant mRNA Stability in Escherichia coli by a Combination of Transcriptional Inducer Wash-Out and qRT-PCR.

Kucharova V, Strand TA, Almaas E, Naas AE, Brautaset T, Valla S - PLoS ONE (2013)

Kinetics of gm-csf and ompA-gm-csf transcript decay, expressed from Pm.The larger plot shows decay kinetics with both gm-csf and ompA-gm-csf transcript levels at time point zero arbitrarily set to one. The smaller plot shown in the upper right corner represents all transcript data relative to the value of gm-csf at time point zero (arbitrarily set to one). Solid lines represent the best fit to the data calculated according to Eqn 4 (Material and Methods). Error bars show the deviation between two biological recurrences. RQ: relative quantification, AU: arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3686738&req=5

pone-0066429-g004: Kinetics of gm-csf and ompA-gm-csf transcript decay, expressed from Pm.The larger plot shows decay kinetics with both gm-csf and ompA-gm-csf transcript levels at time point zero arbitrarily set to one. The smaller plot shown in the upper right corner represents all transcript data relative to the value of gm-csf at time point zero (arbitrarily set to one). Solid lines represent the best fit to the data calculated according to Eqn 4 (Material and Methods). Error bars show the deviation between two biological recurrences. RQ: relative quantification, AU: arbitrary units.
Mentions: In a previous study [15], we showed that expression of the gm-csf gene, encoding human granulocyte-macrophage colony-stimulating factor (GM-CSF), is strongly stimulated at the transcript and protein levels, by fusing the ompA translocation signal sequence in frame with the 5′ end of the gm-csf coding sequence. This therefore represented a different test case for investigation of mRNA stability and its potential role in recombinant protein production. The decay of gm-csf transcript was monitored in parallel for DH5α (pGM29) and DH5α (pGM29ompA) strains. The results showed that the addition of ompA 5′ signal sequence leads to a significant increase in the gm-csf mRNA stability (Figure 4), estimated to be a 4-fold decrease in the relative decay rate; 0.31 for gm-csf (95% CI = 0.30–0.33) and 0.08 for ompA-gm-csf (95% CI: 0.08–0.09).

Bottom Line: The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case.We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system.The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.

ABSTRACT
mRNA stability is one among many parameters that can potentially affect the level of recombinant gene expression in bacteria. Blocking of the entire prokaryotic transcription machinery by addition of rifampicin is commonly used in protocols for analysis of mRNA stability. Here we show that such treatment can be effectively replaced by a simple, non-invasive method based on removal of the relevant transcriptional inducers and that the mRNA decay can then be followed by qRT-PCR. To establish the methodology we first used the m-toluate-inducible XylS/Pm expression cassette as a model system and analyzed several examples of DNA modifications causing gene expression stimulation in Escherichia coli. The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case. To support the experimental data a simple mathematical fitting model was developed to calculate relative decay rates. We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system. The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems. Moreover, as expression systems based on diffusible inducers are almost universally available, the concept can be most likely used to measure mRNA decay for any gene in any cell type that is heavily used in molecular biology research.

No MeSH data available.


Related in: MedlinePlus