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Recombinant Escherichia coli strains with inducible Campylobacter jejuni single domain hemoglobin CHb expression exhibited improved cell growth in bioreactor culture.

Xu L, Xiong W, Yang JK, Li J, Tao XW - PLoS ONE (2015)

Bottom Line: Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells.To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays.Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

View Article: PubMed Central - PubMed

Affiliation: College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China.

ABSTRACT
Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells. Bacterial hemoglobins are oxygen-binding proteins that transfer oxygen from the environment to metabolic processes and allow bacteria to grow even under microaerophilic conditions. To improve the oxygen utilization efficiency of cells and overcome the oxygen shortage in bioreactors, the gene coding for the Campylobacter jejuni single domain hemoglobin (CHb) gene was artificially synthesized and functionally expressed under the control of inducible expression promoters PT7 and Pvgh in Escherichia coli. The effects of the recombinants PT7-CHb and Pvgh-CHb on cell growth were evaluated in aerobic shake flasks, anaerobic capped bottles and a 5-L bioreactor, and a pronounced improvement in cell biomass was observed for CHb-expressing cells. To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays. Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

No MeSH data available.


Related in: MedlinePlus

Semi-quantitative RT-PCR assays of the levels of CHb transcription in four recombinants in the bioreactor.A: Time course of CHb expression visualized by agarose gels with GAPDH used as an internal control; B: The signal intensities of each band were quantified and normalized to those of the corresponding GAPHD band and reported as relative values.
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pone.0116503.g006: Semi-quantitative RT-PCR assays of the levels of CHb transcription in four recombinants in the bioreactor.A: Time course of CHb expression visualized by agarose gels with GAPDH used as an internal control; B: The signal intensities of each band were quantified and normalized to those of the corresponding GAPHD band and reported as relative values.

Mentions: Semi-quantitative RT-PCR was conducted to determine the expression level of the CHb gene under the control of the two promoters. As shown in Fig. 6, the time course expression analysis revealed the constitutive and inducible expression characteristics of the CHb recombinants. For the IPTG inducible expression recombinant PT7-CHb, the highest level of CHb expression was observed in the lag phase and the early logarithmic phase (from 0 h to 10 h). At that point, the relative density of CHb was approximately 1.7-fold greater than that of GAPDH (10 h time point). After that point, the cells entered into the middle stage of the logarithmic phase and then the stationary phase, and the expression level of CHb gradually decreased (Fig. 6A and 6B).


Recombinant Escherichia coli strains with inducible Campylobacter jejuni single domain hemoglobin CHb expression exhibited improved cell growth in bioreactor culture.

Xu L, Xiong W, Yang JK, Li J, Tao XW - PLoS ONE (2015)

Semi-quantitative RT-PCR assays of the levels of CHb transcription in four recombinants in the bioreactor.A: Time course of CHb expression visualized by agarose gels with GAPDH used as an internal control; B: The signal intensities of each band were quantified and normalized to those of the corresponding GAPHD band and reported as relative values.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352031&req=5

pone.0116503.g006: Semi-quantitative RT-PCR assays of the levels of CHb transcription in four recombinants in the bioreactor.A: Time course of CHb expression visualized by agarose gels with GAPDH used as an internal control; B: The signal intensities of each band were quantified and normalized to those of the corresponding GAPHD band and reported as relative values.
Mentions: Semi-quantitative RT-PCR was conducted to determine the expression level of the CHb gene under the control of the two promoters. As shown in Fig. 6, the time course expression analysis revealed the constitutive and inducible expression characteristics of the CHb recombinants. For the IPTG inducible expression recombinant PT7-CHb, the highest level of CHb expression was observed in the lag phase and the early logarithmic phase (from 0 h to 10 h). At that point, the relative density of CHb was approximately 1.7-fold greater than that of GAPDH (10 h time point). After that point, the cells entered into the middle stage of the logarithmic phase and then the stationary phase, and the expression level of CHb gradually decreased (Fig. 6A and 6B).

Bottom Line: Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells.To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays.Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

View Article: PubMed Central - PubMed

Affiliation: College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China.

ABSTRACT
Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells. Bacterial hemoglobins are oxygen-binding proteins that transfer oxygen from the environment to metabolic processes and allow bacteria to grow even under microaerophilic conditions. To improve the oxygen utilization efficiency of cells and overcome the oxygen shortage in bioreactors, the gene coding for the Campylobacter jejuni single domain hemoglobin (CHb) gene was artificially synthesized and functionally expressed under the control of inducible expression promoters PT7 and Pvgh in Escherichia coli. The effects of the recombinants PT7-CHb and Pvgh-CHb on cell growth were evaluated in aerobic shake flasks, anaerobic capped bottles and a 5-L bioreactor, and a pronounced improvement in cell biomass was observed for CHb-expressing cells. To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays. Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

No MeSH data available.


Related in: MedlinePlus