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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

Synthesis of the CHb gene by a two-step gene synthesis method.A: Flow chart depicting the two-step CHb gene synthesis method; B: Agarose gels verifying the PCR product of assembly PCR and overlap extension PCR of the CHb gene.
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pone.0116503.g001: Synthesis of the CHb gene by a two-step gene synthesis method.A: Flow chart depicting the two-step CHb gene synthesis method; B: Agarose gels verifying the PCR product of assembly PCR and overlap extension PCR of the CHb gene.

Mentions: This study used a two-step gene synthesis method [14] to synthesize the single domain Hb gene of C. jejuni (GenBank: KM007077). Briefly, a batch of adjacent oligonucleotides covering both DNA strands of the full length CHb gene was designed using Gene2Oligo software [15] and then synthesized by the solid-phase phosphoramidite method. The sequences of the oligonucleotides are listed in S1 Table. According to the two-step gene synthesis process described in Fig. 1, oligonucleotides were first assembled into two fragments with 15 overlapping bases by assembly PCR, and then these two fragments were assembled into the full length CHb gene by overlap extension PCR. The CHb gene was inserted into the pMD18-T vector (TaKaRa, Dalian, China) and verified by sequencing.


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)

Synthesis of the CHb gene by a two-step gene synthesis method.A: Flow chart depicting the two-step CHb gene synthesis method; B: Agarose gels verifying the PCR product of assembly PCR and overlap extension PCR of the CHb gene.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116503.g001: Synthesis of the CHb gene by a two-step gene synthesis method.A: Flow chart depicting the two-step CHb gene synthesis method; B: Agarose gels verifying the PCR product of assembly PCR and overlap extension PCR of the CHb gene.
Mentions: This study used a two-step gene synthesis method [14] to synthesize the single domain Hb gene of C. jejuni (GenBank: KM007077). Briefly, a batch of adjacent oligonucleotides covering both DNA strands of the full length CHb gene was designed using Gene2Oligo software [15] and then synthesized by the solid-phase phosphoramidite method. The sequences of the oligonucleotides are listed in S1 Table. According to the two-step gene synthesis process described in Fig. 1, oligonucleotides were first assembled into two fragments with 15 overlapping bases by assembly PCR, and then these two fragments were assembled into the full length CHb gene by overlap extension PCR. The CHb gene was inserted into the pMD18-T vector (TaKaRa, Dalian, China) and verified by sequencing.

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