Limits...
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

Cultivation parameters and growth conditions of the recombinant cells in the 5-L bioreactor.A: The cultivation parameters; B: The control cells carrying the empty pUC18 vector; C: The recombinant PT7-CHb; D: The recombinant Pvgh-CHb. E. coli cells carrying blank vectors were used as the controls. Seed cells of the recombinants were inoculated at a 1:100 ratio into LB medium containing 50 mg/ml ampicillin for Pvgh-CHb and 20 mg/mL tetracycline for PT7-CHb. For recombinant PT7-CHb, 0.05 mM IPTG was added into the medium to induce CHb expression. The culture temperature was kept at 37 ∞C, and the pH value of the medium was kept at pH 6.8 with 200 mM NaOH titration. Before inoculation, LB liquid was injected into sterile air for 30 min, and then the dissolved oxygen (DO) of the medium was set as 100%. The error bars indicated the standard deviation generated from three replicates data. * indicates the significant differecne (P<0.05) at 18 h time point calculated by one-way ANOVA analysis.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4352031&req=5

pone.0116503.g005: Cultivation parameters and growth conditions of the recombinant cells in the 5-L bioreactor.A: The cultivation parameters; B: The control cells carrying the empty pUC18 vector; C: The recombinant PT7-CHb; D: The recombinant Pvgh-CHb. E. coli cells carrying blank vectors were used as the controls. Seed cells of the recombinants were inoculated at a 1:100 ratio into LB medium containing 50 mg/ml ampicillin for Pvgh-CHb and 20 mg/mL tetracycline for PT7-CHb. For recombinant PT7-CHb, 0.05 mM IPTG was added into the medium to induce CHb expression. The culture temperature was kept at 37 ∞C, and the pH value of the medium was kept at pH 6.8 with 200 mM NaOH titration. Before inoculation, LB liquid was injected into sterile air for 30 min, and then the dissolved oxygen (DO) of the medium was set as 100%. The error bars indicated the standard deviation generated from three replicates data. * indicates the significant differecne (P<0.05) at 18 h time point calculated by one-way ANOVA analysis.

Mentions: To test whether the recombinant E. coli expressing hemoglobin CHb could improve the cell growth in the bioreactor or not, we recorded the growth curves of four recombinants in a 5-L bench top bioreactor (Fig. 5). Due to better temperature, pH and aeration control (Fig. 5A), the recombinant cells grown better in bioreactor than in the flasks and capped bottles. As shown by the control cells, the cell density reached and the wet cell weight reached OD600 = 2.2 and 14.0 g/L, respectively, apparently higher than those in the flasks (Fig. 3) and capped bottles (Fig. 4).


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)

Cultivation parameters and growth conditions of the recombinant cells in the 5-L bioreactor.A: The cultivation parameters; B: The control cells carrying the empty pUC18 vector; C: The recombinant PT7-CHb; D: The recombinant Pvgh-CHb. E. coli cells carrying blank vectors were used as the controls. Seed cells of the recombinants were inoculated at a 1:100 ratio into LB medium containing 50 mg/ml ampicillin for Pvgh-CHb and 20 mg/mL tetracycline for PT7-CHb. For recombinant PT7-CHb, 0.05 mM IPTG was added into the medium to induce CHb expression. The culture temperature was kept at 37 ∞C, and the pH value of the medium was kept at pH 6.8 with 200 mM NaOH titration. Before inoculation, LB liquid was injected into sterile air for 30 min, and then the dissolved oxygen (DO) of the medium was set as 100%. The error bars indicated the standard deviation generated from three replicates data. * indicates the significant differecne (P<0.05) at 18 h time point calculated by one-way ANOVA analysis.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116503.g005: Cultivation parameters and growth conditions of the recombinant cells in the 5-L bioreactor.A: The cultivation parameters; B: The control cells carrying the empty pUC18 vector; C: The recombinant PT7-CHb; D: The recombinant Pvgh-CHb. E. coli cells carrying blank vectors were used as the controls. Seed cells of the recombinants were inoculated at a 1:100 ratio into LB medium containing 50 mg/ml ampicillin for Pvgh-CHb and 20 mg/mL tetracycline for PT7-CHb. For recombinant PT7-CHb, 0.05 mM IPTG was added into the medium to induce CHb expression. The culture temperature was kept at 37 ∞C, and the pH value of the medium was kept at pH 6.8 with 200 mM NaOH titration. Before inoculation, LB liquid was injected into sterile air for 30 min, and then the dissolved oxygen (DO) of the medium was set as 100%. The error bars indicated the standard deviation generated from three replicates data. * indicates the significant differecne (P<0.05) at 18 h time point calculated by one-way ANOVA analysis.
Mentions: To test whether the recombinant E. coli expressing hemoglobin CHb could improve the cell growth in the bioreactor or not, we recorded the growth curves of four recombinants in a 5-L bench top bioreactor (Fig. 5). Due to better temperature, pH and aeration control (Fig. 5A), the recombinant cells grown better in bioreactor than in the flasks and capped bottles. As shown by the control cells, the cell density reached and the wet cell weight reached OD600 = 2.2 and 14.0 g/L, respectively, apparently higher than those in the flasks (Fig. 3) and capped bottles (Fig. 4).

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