Hydrogen production associated protein, coding genes thereof and application thereof
A technology encoding genes and proteins, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of no reports of genes, and achieve the effects of strong substrate utilization, wide substrate range and strong environmental adaptability.
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Embodiment 1
[0055] Embodiment 1, the acquisition of Enterobacter aerogenes ldhd gene
[0056] According to the published D-lactate dehydrogenase (D-lactate dehydrogenase) gene sequence, a pair of degenerate primers Ldh-fw and Ldh-rw were designed. PCR amplification was performed using the genomic DNA of Enterobacter aerogenes (E. aerogenes IAM1183) (purchased from the Institute of Applied Microbiology (IAM) strain bank, University of Tokyo, Japan) as a template.
[0057] PCR products were detected by agarose gel electrophoresis. figure 1 . figure 1 Middle, 1: PCR product; 2: Marker.
[0058] The results showed that a band with a molecular weight of about 900bp was obtained. The PCR product was stored at 4°C and sent to Shanghai Invitrogen Corporation for sequencing. The sequencing results showed that the amplified nucleotide sequence was shown in sequence 2 in the sequence listing, and the encoded amino acid sequence was the protein in sequence 1 in the sequence listing.
[0059] Acco...
Embodiment 2
[0060] Example 2, the acquisition of Enterobacter aerogenes E.aerogenes IAM1183-Δldhd
[0061] 1. Construction of suicide vector pGP704-ldhd
[0062] A pair of primers Ldh-E-fw and Ldh-E-rw with EcoR I restriction sites were designed. Using the genomic DNA of Enterobacter aerogenes (E. aerogenes IAM1183) as a template for PCR amplification, a fragment of more than 540 bp was obtained. EcoRI digestion PCR product, pGP704 [Kolter, R.Inuzuka, M. and Helinski, D. (1978) with EcoR I digestion. Cell 15: 1199-1208, Miller, V. and Mekalanos, J. (1988) .J.Bact.170:2575-2583] Connected. The ligation product was transformed into E.coli DH5α (Dalian Bao Biological Co., Ltd.), screened on LB chloramphenicol (24mg / mL) resistance plate, picked the correct transformants, and extracted the plasmid for PCR identification, as follows:
[0063] A pair of primers, Primer-Pgp-1-fw and Primer-Pgp-2-rw, were designed according to the sequence on pGP704. The recombinant plasmid was identified by P...
Embodiment 3
[0071] Embodiment 3, growth characteristics, hydrogen production performance and metabolic flow of engineering bacteria
[0072] 1. Growth characteristics, hydrogen production performance and metabolic flux of engineered bacteria under shake flask culture conditions
[0073] The engineering bacteria E.aerogenes IAM1183-Δldhd and the wild bacteria E.aerogenes IAM1183 were cultured in shake flasks respectively, and their growth characteristics, hydrogen production performance and metabolic flux were tested. The specific steps are as follows:
[0074] Put 20mL of glucose medium in a 70mL serum bottle, insert the activated engineered bacteria into the medium, and carry out shake flask culture; at the same time, carry out shake flask culture with the same conditions for the activated wild bacteria as a control.
[0075] (1) Seed culture: inoculate activated engineered bacteria (or wild bacteria) into a 15ml centrifuge tube containing 5ml LB medium, and culture overnight at 37°C, 17...
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