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Recombinant escherichia coli and hydrogen production application

A technology for recombining Escherichia coli and Escherichia coli, used in applications, recombinant DNA technology, bacteria, etc.

Active Publication Date: 2022-06-03
CHENGDU UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key to hydrogen production by anaerobic fermentation is that the target strain has a relatively high hydrogen production efficiency, but simple strain selection cannot meet the current demand

Method used

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  • Recombinant escherichia coli and hydrogen production application
  • Recombinant escherichia coli and hydrogen production application
  • Recombinant escherichia coli and hydrogen production application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Construction of recombinant Escherichia coli containing [FeFe]-hydrogenase gene and phosphofructokinase isoenzyme I gene

[0019] 1. Resistance test

[0020] Escherichia coli BL21 (DE3) were streaked on LB solid plates of commonly used antibiotics (such as Amp, Kana, Chl and Str), cultured overnight in a constant temperature incubator at 37°C, and the next day was observed for colony growth, such as Some are judged to be insensitive to the antibiotic, and vice versa. Escherichia coli insensitive to antibiotics kana and Str were obtained.

[0021] 2. Construction of CRISPR-B Plasmid

[0022] Taking Chlamydomonas reinhardtii [FeFe]-hydrogenase gene hyd1 as the main body, using araBAD promoter to drive the expression of hyd1 gene, using two terminators rrnB T1terminator and rrnB T2 terminator to terminate the expression of hyd1 gene . like figure 1 shown in KI. The final knock-in nucleotide sequence is shown in SEQ ID No.1. The knock-in sequence and the ho...

Embodiment 2

[0042] Example 2 Application of Recombinant Escherichia coli Hydrogen Production

[0043] A solid medium was prepared, and agar powder was added to the LB medium containing 50 μg / mL karamycin to prepare a solid medium. The thawed activated recombinant E. coli was inoculated into LB solid medium. Incubate overnight in a 37°C incubator.

[0044] The single colony recombinant Escherichia coli grown in LB solid medium was inoculated into LB medium containing 50 μg / mL karamycin, and expanded in a constant temperature incubator at 37°C.

[0045]The expanded recombinant E. coli was inoculated into 200 ml of hydrogen-producing medium containing 50 μg / mL karamycin. The hydrogen-producing medium contained 1.6 parts tryptone, 1 part yeast extract and 0.5 part sodium chloride, and 2 parts sucrose was added. , the pH value of the hydrogen production medium was 6.0, and 27 mL of hydrogen was generated within 8-48 h of incubation in a constant temperature incubator at 37 °C.

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Abstract

The invention discloses recombinant escherichia coli and a hydrogen production application thereof. The recombinant escherichia coli is obtained by transferring a [FeFe]-hydrogenase gene hyd1, which is derived from Chlamydomonas reinhardtii and is shown in SEQ ID NO.1, and a phosphofructokinase isoenzyme I gene pfkA, which is shown in SEQ ID NO.2, into a DNA (Deoxyribonucleic Acid) chromosome of the escherichia coli. The hydrogen-producing recombinant escherichia coli screened by the invention can efficiently produce hydrogen in cells, and takes glucose, fructose, cane sugar, starch and glycerol as substrates. The recombinant Escherichia coli strain for screening hydrogen production has the advantages that inducers such as IPTG (isopropyl-beta-d-thiogalactoside) are not added, interference of high-concentration glucose can be overcome, cost reduction is brought to green hydrogen preparation, and the recombinant Escherichia coli strain is environment-friendly, has practical value and can realize'safe, clean, economical and continuous' large-scale production of green energy.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli containing [FeFe]-hydrogenase gene and phosphofructokinase isoenzyme I gene on chromosome and its hydrogen production application, and belongs to the technical field of microbial hydrogen production. Background technique [0002] Hydrogen is a clean energy, but traditional hydrogen is produced from non-renewable fossil energy such as coal and oil, which consumes a lot of energy in addition to causing environmental pollution. Renewable energy production "green hydrogen" technology represented by biological hydrogen production is considered to be one of the most ideal energy solutions in the future. Biological hydrogen production is the use of biological metabolism to convert water, organic waste or biological substrates into hydrogen, which reduces energy consumption and does not pollute in the production process. It is an important direction of "green hydrogen" research. [0003] The development ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/70C12P3/00C12R1/19
CPCC12N9/0067C12N9/1205C12Y112/01002C12Y207/01011C12N15/70C12P3/00Y02E60/36
Inventor 王以明朱鹏程童晋盛钧超杨旭
Owner CHENGDU UNIVERSITY OF TECHNOLOGY
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