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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

The nucleotide sequences of the promoters synthesized in this study.Italics indicate the restriction sites, and bolded letters are the conserved motifs of the promoters. Underlined nucleotides is a FNR-binding site, and dash line indicated is a portion of cAMP-CAP binding site.
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pone.0116503.g002: The nucleotide sequences of the promoters synthesized in this study.Italics indicate the restriction sites, and bolded letters are the conserved motifs of the promoters. Underlined nucleotides is a FNR-binding site, and dash line indicated is a portion of cAMP-CAP binding site.

Mentions: The hypoxia-inducible promoter Pvgh of Vitreoscilla [16] was synthesized using the same process described above. The promoter sequences are listed in Fig. 2, and the oligonucleotides used to synthesize these promoters are listed in S2 Table. The IPTG-inducible expression recombinant PT7-CHb was constructed by inserting the CHb gene into the pET28a vector at the NdeI and XhoI sites and then transfected into E. coli BL21 (DE3) cells. Hypoxia-inducible recombinant Pvgh-CHb was constructed by fusing Pvgh with the CHb gene by NdeI sticky ends to form Pvgh-CHb fragments. These fragments were then inserted into the pUC18 vector at the BamHI and XhoI sites and transfected into the E. coli DH5α strain.


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)

The nucleotide sequences of the promoters synthesized in this study.Italics indicate the restriction sites, and bolded letters are the conserved motifs of the promoters. Underlined nucleotides is a FNR-binding site, and dash line indicated is a portion of cAMP-CAP binding site.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116503.g002: The nucleotide sequences of the promoters synthesized in this study.Italics indicate the restriction sites, and bolded letters are the conserved motifs of the promoters. Underlined nucleotides is a FNR-binding site, and dash line indicated is a portion of cAMP-CAP binding site.
Mentions: The hypoxia-inducible promoter Pvgh of Vitreoscilla [16] was synthesized using the same process described above. The promoter sequences are listed in Fig. 2, and the oligonucleotides used to synthesize these promoters are listed in S2 Table. The IPTG-inducible expression recombinant PT7-CHb was constructed by inserting the CHb gene into the pET28a vector at the NdeI and XhoI sites and then transfected into E. coli BL21 (DE3) cells. Hypoxia-inducible recombinant Pvgh-CHb was constructed by fusing Pvgh with the CHb gene by NdeI sticky ends to form Pvgh-CHb fragments. These fragments were then inserted into the pUC18 vector at the BamHI and XhoI sites and transfected into the E. coli DH5α strain.

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