CN108220319A - There is the carrier pET32a-fdhF-1 for producing hydrogen functional gene and its structure and application in Escherichia coli - Google Patents

There is the carrier pET32a-fdhF-1 for producing hydrogen functional gene and its structure and application in Escherichia coli Download PDF

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CN108220319A
CN108220319A CN201810093583.5A CN201810093583A CN108220319A CN 108220319 A CN108220319 A CN 108220319A CN 201810093583 A CN201810093583 A CN 201810093583A CN 108220319 A CN108220319 A CN 108220319A
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fdhf
pet32a
escherichia coli
gene
hydrogen
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王海燕
刘伟
赵雅琪
徐岚婷
王冕超
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Beijing University of Technology
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    • C12Y102/01002—Formate dehydrogenase (1.2.1.2)

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Abstract

In Escherichia coli there is the carrier pET32a fdhF 1 of production hydrogen functional gene and its build and apply, be related to gene engineering technology field.1 genophores of pET32a fdhF are the pET32a plasmids for including e. coli bl21 hydrogenlyase fdhF genes.Its structure is the fdhF genetic fragments by designing the hydrogenlyase of primer amplification e. coli bl21 (formate dehydrogenase H), and pass through in the pET32a carriers that T4 DNA ligases are connected to after digestion, it is named as pET32a fdhF 1, gene sequencing is carried out, verifies the integrality of gene.1 plasmids of pET32a fdhF are transformed into Escherichia coli, culture production hydrogen is carried out in hydrogen culture medium is produced.Escherichia coli hydrogen generation efficiency can be improved by being transferred to carrier pET32a fdhF 1.

Description

There is the carrier pET32a-fdhF-1 and its structure of production hydrogen functional gene in Escherichia coli It builds and applies
Technical field
The present invention relates to gene engineering technology field, more particularly to a kind of production hydrogen functional gene carrier pET32a-fdhF-1 And its it builds and applies.
Background technology
Fossil energy gradually decreases at present, and in new energy field, Hydrogen Energy is as a kind of free of contamination clean energy resource And energy carrier, in recent years its development and utilization technology obtain the great attention of industrialized country.Instantly hydrogen gas production mainly has Fossil energy converts, three kinds of modes of decomposition and biological hydrogen production of water.First two is widely used, but pollutes environment and do not have Sustainable developability.Biological hydrogen production has safe, inexpensive, environmental-friendly and renewable physical resources is rationally utilized excellent Point, if can extensive use, important meaning is had to environmental protection.It is trained since Escherichia coli can produce hydrogen microorganism with respect to other Foster at low cost and realizability is strong, therefore is the optimal selection for developing hydrogen production by biological.Wild Escherichia coli do not produce hydrogen or production hydrogen Less, the existing Escherichia coli being transformed by genetic engineering are unstable and hydrogen generation efficiency is low, it would be highly desirable to outer by being artificially introduced Carrying out gene makes its hydrogen generation efficiency increase, to meet the needs of human development hydrogen energy source.
Invention content
The object of the present invention is to provide a production hydrogen functional gene carrier pET32-fdhF-1 and its construction methods.It can It applied in e. coli bl21, can be improved the hydrogen generation efficiency of Escherichia coli.
In order to solve the above technical problems, the technical solution used in the present invention is：
The present invention provides a kind of production hydrogen functional gene carrier pET32a-fdhF-1, is the formic acid for including Escherichia coli The pET32a-fdhF-1 plasmids of dehydrogenase gene fdhF.(explanation：By formate dehydrogenase gene fdhF progress gram in Escherichia coli Grand pioneering for the present invention for improving hydrogen output, selection pET32a is that carrier expression formate dehydrogenase gene fdhF is also this hair Bright innovation)
The present invention also provides the construction method of production hydrogen functional gene carrier pET32a-fdhF-1, mainly by using limit I digestion PCR products of property restriction endonuclease Nde I and Xho processed and plasmid reuse both T4DNA ligases connections.
(explanation：For improve digesting efficiency, can PCR product be first subcloned into pCloneEZ-TOPO carriers, then again from turn Plasmid and digestion are extracted in the bacterium of change.)
Specific operation process includes the following steps：(1) prepared by DNA fragmentation：With reference to the digestion position on expression vector pET32a Point Nde I and XhoI design the primer of formate dehydrogenase gene fdhF in Escherichia coli, and use high-fidelity DNA polymerase PCR reaction amplification fdhF genes.PCR product is subcloned into pCloneEZ-TOPO carriers, is then carried from the bacterium of conversion again Take plasmid and with Nde I and XhoI digestions, gel extraction fdhF genetic fragments.
(2) prepared by linearized vector：The use of pET32a is carrier, obtaining linearisation using Nde I and XhoI double digestion carries Body.
(3) coupled reaction：By T4DNA ligases, ready linearized vector has been connected with fdhF genetic fragments Come, obtain recombinant vector.
(4) it converts：The recombinant vector built is transferred in competence bacillus coli DH 5 alpha.
(5) identification of positive transformant：Picked clones bacterium using the primer used in amplification fdhF genes, carries out bacterium colony PCR Reaction, is identified by electrophoresis, after determining positive transformant, then extracts plasmid, sequencing.
The present invention also provides the applications of production hydrogen functional gene carrier pET32a-fdhF-1：By pET32a-fdhF-1 plasmids It is transformed into e. coli bl21, is cultivated in hydrogen culture medium is produced and carry out production hydrogen.(explanation：Selection e. coli bl21 is host The plasmid of structure is transformed into this bacterium to produce hydrogen reaction and the innovation of the present invention by bacterium.)
Bacillus coli DH 5 alpha used in the present invention and BL21 (DE3) bacterial strain are purchased from Tiangeng biochemical technology (Beijing) limited public affairs Department.
The beneficial effects of the invention are as follows pET32a-fdhF-1 can be applied in Escherichia coli, can improve Escherichia coli production Hydrogen efficiency.
The present invention also provides production hydrogen functional gene carrier pET32a-fdhF-1 destination protein expression condition, due to There are one selenocysteine codon UGA in fdhF genes, are typically used as terminator codon.When cell growth lacks selenium, The translation of selenoprotein can stop at UGA codons, become imperfect without the albumen of function.And add in SeO3 2-It can promote UGA translates into selenocysteine so that destination protein is expressed.(explanation：Add in SeO3 2-Make the first that pET32a is carrier Dehydrogenase gene fdhF be expressed as of the invention pioneering)
Description of the drawings
The invention will be further described below in conjunction with the accompanying drawings：
Fig. 1 is the constructing plan schematic diagram of carrier pET32a-fdhF-1；
The western-blot of hydrogenlyase is analyzed when Fig. 2 is using glucose as substrate cultivation, wherein recombinant protein At 80.4kDa, protein content is proportionate fdhF with band gray value；
As shown in Figure 2, when using glucose as substrate, the expressing quantity of pET32a-fdhF-1 transformed bacterias is higher than The expression quantity of pET32a transformed bacterias.Swimming lane 2,3 has faint band, and swimming lane 1 does not have band, and reason may be that the bacterial strain of picking is lived Property is weaker；Swimming lane 4,5,6 is generated without band, and the albumen for illustrating not recombinate is not expressed；
The western-blot of hydrogenlyase is analyzed when Fig. 3 is using sodium formate as substrate cultivation, wherein recombinant protein At 80.4kDa, protein content is proportionate fdhF with band gray value；
From the figure 3, it may be seen that when using sodium formate as substrate cultivation, the expression quantity of pET32a-fdhF-1 transformed bacterias is higher than The expression quantity of pET32a transformed bacterias.Swimming lane 1,2 has band generation, illustrates that recombinant protein fdhF is expressed, swimming lane 3 is produced without band Raw, reason may be that the strain activity of picking is weaker；Swimming lane 4,5,6 is generated without band, and the albumen for illustrating not recombinate does not have table It reaches；
Fig. 4 western-blot electrophoretograms under the conditions of different substrate cultivations, wherein recombinant protein fdhF is in 80.4kDa Place, protein content are proportionate with band gray value；
As shown in Figure 4, swimming lane 1, in 2,3 band gray value and brightness be more than 4,5,6 band gray value of swimming lane.Illustrate not With under the condition of culture of substrate, the expression quantity of recombinant protein fdhF is higher than using glucose the bottom of as when using sodium formate as substrate cultivation The expression quantity of recombinant protein fdhF during object culture.
Fig. 5 adds in SeO3 2-Under condition of culture western-blot analyze, wherein recombinant protein fdhF at 80.4kDa, Protein content is proportionate with strip width；
As shown in Figure 5, swimming lane 1, band gray value is more than 3,4 band gray value of swimming lane in 2, and swimming lane 5,6,7,8 exists Occur at 80.4kDa without band.Illustrate, using sodium formate as substrate cultivation, adding in SeO3 2-Recombinant protein fdhF during culture Expression quantity higher than not adding in SeO3 2-When recombinant protein fdhF expression quantity, while SeO3 2-It cannot make the egg of pET32a transformed bacterias It is expressed in vain；
Swimming lane 1,2：PET32a-fdhF-1 transformed bacterias, containing 6 μm of ol/L SeO3 2-, 2 Duplicate Samples
Swimming lane 3,4：PET32a-fdhF-1 transformed bacterias, without SeO3 2-, 2 Duplicate Samples
Swimming lane 5,6：PET32a transformed bacterias, containing 6 μm of ol/L SeO3 2-, 2 Duplicate Samples
Swimming lane 7,8：PET32a transformed bacterias, without SeO3 2-, 2 Duplicate Samples
Formate dehydrogenase enzymatic activity when table 1 is using sodium formate or glucose as substrate
It can be obtained by table 1, the recombinant protein fdhF activity when pET32a-fdhF-1 transformed bacterias are using sodium formate as substrate cultivation Recombinant protein fdhF activity during higher than using glucose as substrate cultivation, recombinant protein fdhF activity improve about 30%.PET32a turns Change bacterium itself and also contain formate dehydrogenase gene, therefore pET32a transformed bacterias have certain background values.
Table 2SeO3 2-Influence to formate dehydrogenase enzymatic activity
It can be obtained by table 2, when pET32a-fdhF-1 transformed bacterias add in SeO3 2-Recombinant protein fdhF activity is not higher than during culture Add in SeO3 2-When recombinant protein fdhF activity, improve about 30%.
PET32a-fdhF-1 transformed bacterias hydrogen-producing characteristic when Fig. 6 is using glucose as substrate cultivation；
As seen from Figure 6, pET32a-fdhF-1 transformed bacterias accumulation hydrogen output is consistently higher than when using glucose as substrate Compare bacterium (pET32a transformed bacterias), and difference is maximum both after accumulative production hydrogen 4 hours.
PET32a-fdhF-1 transformed bacterias hydrogen-producing characteristic when Fig. 7 is using sodium formate as substrate cultivation；
As seen from Figure 7, pET32a-fdhF-1 transformed bacterias accumulation hydrogen output is consistently higher than when using sodium formate as substrate Bacterium (pET32a transformed bacterias) is compareed, the hydrogen-producing speed of pET32a-fdhF-1 transformed bacterias is gradually increased, both after inducing 5 hours Difference is maximum.
Specific embodiment
Below in conjunction with the embodiment and attached drawing in the present invention, the technical solution in the embodiment of the present invention is carried out it is clear, It is fully described by, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Base Embodiment in the present invention, those of ordinary skill in the art obtained without making creative work it is all its His embodiment, shall fall within the protection scope of the present invention.
Embodiment 1：
(1) it searches and closes on NCBI (National Center for Biotechnology Information) website In the gene order of E. coli formate dehydrogenase, in conjunction with pET32a plasmid sequences, using DNAMAN softwares, design and carry The primer of the formate dehydrogenase gene of restriction endonuclease sites.Give designed primer to raw work bioengineering (Shanghai) stock Part Co., Ltd carries out primer synthesis.
The primer designed is：
Sense primer Forward-Primer：5’-CATATGAAAAAAGTCGTCACGGTTTGC-3’
Downstream primer Reverse-Primer：5’-CTCGAGCGCCAGTGCCGCTTCGCG-3’
(2) according to designed primer, fdhF genetic fragments are expanded using round pcr.PCR condition settings：94 DEG C of denaturation 30s, 58 DEG C of 30s of renaturation extend 72 DEG C of 3min, 30 cycles；50 μ L systems：1 μ L genome of E.coli DNA, 1 μ L upstreams are drawn Object (10 μm of ol/L), 1 μ L downstream primers (10 μm of ol/L), 5 μ 10 × Pfu of L Buffer, 4 μ LdNTP Mix (2.5 μm of ol/L), 1 μ LPfu enzymes, ddH2O polishings are to 50 μ L.(pfu archaeal dna polymerases are purchased from TIANGEN Biotech (Beijing) Co., Ltd.)
(3) PCR product is subcloned into pCloneEZ-TOPO carriers (clone smarter technologies), so Extract plasmid from the bacterium of conversion again afterwards and with I double digestion of Nde I and Xho.
(4) digestion products with agarose gel electrophoresis are detached, and fdhF genetic fragments is cut (about under gel imager 2100bp), it is recycled with plastic recovery kit (Omega Bio-Tek, USA), obtains the about 30 μ L of fdhF genetic fragments after digestion.
(5) using I double digestion pET32a carriers of restriction enzyme Nde I and Xho.It is detached with agarose gel electrophoresis, and The large fragment (about 5400bp) of cut vector, is recycled with plastic recovery kit under gel imager, obtains linearized vector about 30μL。
(6) the pET32a carriers of linearisation are obtained from step (5), step (4) obtains fdhF genes, obtained through coupled reaction PET32a plasmid 20uL containing fdhF genetic fragments, and convert.Process is as follows：1. coupled reaction system is 1 μ L pET32a matter Grain, 3 μ L fdhF genetic fragments, 1 μ L T4ligase, 2 μ 10 × Ligation of L Buffer, ddH2O complements to 20 μ L.② 16 DEG C of reaction 10h；3. 5 μ L is taken to add in the DH5 α competence of 100 μ L the plasmid connected, 15min is placed in ice；④42℃ Water-bath 30s, then 2min is placed in ice；5. add in 300 μ L LB culture mediums (without antibiotic), 37 DEG C of 200rpm shaken cultivations 60min；6. 200 μ L is taken to be applied to the solid LB media plate of ammonia benzyl resistance, 37 DEG C of overnight incubations.
(7) identification (bacterium colony PCR methods) of positive transformant：It is 1. fully mixed in picking monoclonal bacterium colony to 10 μ L sterile waters It closes, the template that 0.5 μ L mixed liquors is taken to be reacted as PCR；2. primer be expand fdhF genes used in primer 3. PCR react item Part：94 DEG C of 30s, 58 DEG C of 30s of renaturation are denaturalized, extend 72 DEG C of 3min, 30 cycles；4. take 10 μ L PCR product electroresis appraisals；⑤ After confirming transformant, remaining 8 μ L bacterium solutions are transferred to LB culture mediums (antibiotic of benzyl containing ammonia), 37 DEG C of overnight incubations；Finally use Plasmid extraction kit (OmegaBio-Tek, USA) extracts plasmid.
(8) plasmid in (7) is sent to genome company's sequencing, to verify the integrality of gene；
(9) plasmid after being had verified that in (8) is transformed into e. coli bl21 (DE3), is examined using hydrogen culture medium is produced Its hydrogen-producing characteristic.
It is cultivated in the 4mL LB culture mediums containing 100 μ g/ml ammonia benzyls, condition of culture：37 DEG C, 200rpm is incubated overnight, Recombination bacillus coli after being enriched with；
By the recombination bacillus coli in LB culture mediums by the amount of 1%v/v be inoculated into containing respectively contain 0.1mol/L grapes It ferments in the sterile test tube of the 10mL of sugar or sodium formate production hydrogen culture mediums, fermentation condition：1. 37 DEG C, 180rpm cultures to glucose For substrate OD600nm=0.8, sodium formate is substrate OD600nm=0.6；2. IPTG derivants are added in a concentration of 0.3mmol/ L, 37 DEG C, 180rpm.After IPTG is added in, per the gaseous sample for extracting 100uL from anaerobism bottle every other hour, GC3420 gas is used Chromatography carries out gas component and concentration analysis.Analysis condition, the 5A molecular sieves of 80/100 mesh, TCD detector temperatures 120 DEG C, 80 DEG C of injector temperature, 80 DEG C of column temperature, carrier gas is high pure nitrogen.Production hydrogen experimental period continues 5 hours altogether.
After production hydrogen experiment carries out 5 hours, hydrogen curve is produced as shown in Figure 6 and Figure 7, the e. coli bl21 with not producing hydrogen (DE3) compare, pET32a-fdhF-1 can make the Escherichia coli for not producing hydrogen generate hydrogen, and work well.Experiment also turns out, this The constructed production hydrogen plasmid pET32a-fdhF-1 of invention can be used to improve hydrogen-producing bacteria, improve hydrogen generation efficiency.It is by sodium formate Recombination bacillus coli production hydrogen effect is better than using glucose as the recombination bacillus coli of substrate during substrate.Conversion bacterial species are only limitted to The prokaryotic micro-organisms of hydrogen is produced by ferronickel hydrogen enzyme.
The Escherichia coli that IPTG is induced carry out Western Blot detections using anti-His antibody.IPTG can be lured Recombinant protein expression is led, and pillar location meets expected (theoretical value 80.4kDa).
The Escherichia coli that IPTG is induced measure enzyme activity.Measure the microorganism formate dehydrogenase (FDH) that enzyme activity uses ELISA KIT kits.Enzyme activity determination step is as follows：1. taking the bacterium solution of 2h after 2mL inductions, 13000rpm centrifugation 2min remove Supernatant adds in the processing of 1ml PBS ultrasonications；2. in 48 orifice plates, standard items set 5 concentration points, 10 holes, each concentration Parallel hole is set, adds in the standard items of 50 μ L various concentrations, blank well sets a hole and adds in 50 μ L distilled water, sample to be tested Hole first adds 40 μ L of sample diluting liquid in sample to be tested hole on enzyme mark coating plate, then adds 10 μ L of sample to be tested again；3. use sealing plate 37 DEG C of film sealing plate postposition incubates 30 minutes；4. taking sealing plate film off, liquid is abandoned, cleaning solution is filled it up in drying per hole, after standing 30 seconds It discards, is so repeated 5 times, pat dry；3. 4. 5. PBS adds in 50 μ L of enzyme marking reagent per hole, except blank well, repetitive operation；It is 6. every Hole first adds in color developing agent A50 μ L, adds color developing agent B50 μ L, and gently concussion shakes up, and 37 DEG C are protected from light colour developing 15min；7. add per hole 50 μ L of terminate liquid terminate reaction；8. being returned to zero with blank well, 450nm wavelength sequentially measures each hole absorbance.Measurement result is shown in Table 1, Table 2.
It can be that professional and technical personnel in the field realize or use that the above embodiment, which is intended to illustrate the present invention, to above-mentioned Embodiment is modified and be will be apparent for those skilled in the art, therefore the present invention includes but not limited to The above embodiment, it is any to meet the claims or specification description, meet with principles disclosed herein and novelty, The method of inventive features, technique, product, each fall within protection scope of the present invention.
Influence of the different substrates of table 1 to formate dehydrogenase enzymatic activity
Table 2SeO3 2-Influence to formate dehydrogenase enzymatic activity
Note：Using sodium formate as substrate
FdhF gene orders (sequencing result after recombination) in Escherichia coli
Sense primer Forward-Primer：5’-catatgaaaaaagtcgtcacggtttgc-3’
Downstream primer Reverse-Primer：5’-ctcgagcgccagtgccgcttcgcg-3’
1 atgaaaaaag tcgtcacggt ttgcccctat tgcgcatcag gttgcaaaat caacctggtc
61 gtcgataacg gcaaaatcgt ccgggcggag gcagcgcagg ggaaaaccaa ccagggtacc
121 ctgtgtctga agggttatta tggctgggat ttcattaacg atacccagat cctgaccccg
181 cgcctgaaaa cccccatgat ccgtcgccag cgtggcggca aactcgaacc tgtttcctgg
241 gatgaggcac tgaattacgt tgccgagcgc ctgagcgcca tcaaagagaa gtacggtccg
301 gatgccatcc agacgaccgg ctcctcgcgt ggtacgggta acgaaaccaa ctatgtaatg
361 caaaaatttg cgcgcgccgt tattggtacc aataacgttg actgctgcgc tcgtgtctga
421 cacggcccat cggttgcagg tctgcaccaa tcggtcggta atggcgcaat gagcaatgct
481 attaacgaaa ttgataatac cgatttagtg ttcgttttcg ggtacaaccc ggcggattcc
541 cacccaatcg tggcgaatca cgtaattaac gctaaacgta acggggcgaa aattatcgtc
601 tgcgatccgc gcaaaattga aaccgcgcgc attgctgaca tgcacattgc actgaaaaac
661 ggctcgaaca tcgcgctgtt gaatgcgatg ggccatgtca ttattgaaga aaatctgtac
721 gacaaagcgt tcgtcgcttc ccgtacagaa ggctttgaag agtatcgtaa aatcgttgaa
781 ggctacacgc cggagtcggt tgaagatatc accggcgtca gcgccagtga gattcgtcag
841 gcggcacgga tgtatgccca ggcgaaaagc gccgccatcc tgtggggcat gggtgtaacc
901 cagttctacc agggcgtgga aaccgtgcgt tctctgacca gcctcgcgat gctgaccggt
961 aacctcggta agccgcatgc gggtgtaaac ccggttcgtg gtcagaacaa cgtgcagggt
1021 gcctgcgata tgggcgcgct gccggatacg tatccgggat accagtacgt gaaagatccg
1081 gctaaccgcg agaaattcgc caaagcctgg ggcgtggaaa gcctgcctgc tcataccggt
1141 tatcgcatca gcgagctgcc gcaccgcgca gcgcatggcg aagtgcgtgc cgcgtacatt
1201 atgggcgaag atccgctgca aactgacgcg gagctgtcgg cagtacgtaa agcctttgaa
1261 gatctggaac tggttatcgt tcaggacatc tttatgacca aaaccgcgtc ggcggcggat
1321 gtcattttgc cgtcaacgtc gtggggcgag catgaaggcg tgtttactgc ggctgaccgt
1381 ggcttccagc gtttcttcaa agcagttgaa ccgaaatggg atctgaaaac ggactggcaa
1441 atcatcagtg aaatcgccac ccgtatgggt tatccgatgc actacaacaa cacccaggag
1501 atctgggatg agttgcgtca tctgtgcccg gatttctacg gtgcgactta cgagaaaatg
1561 ggcgaactgg gcttcattca gtggccttgc cgcgatactt cagatgccga tcaggggact
1621 tcttatctgt ttaaagagaa gtttgatacc ccgaacggtc tggcacagtt cttcacctgc
1681 gactgggtag cgccaatcga caaactcacc gacgagtacc cgatggtact gtcaacggtg
1741 cgtgaagttg gtcactactc ttgccgttcg atgaccggta actgtgcggc actggcggcg
1801 ctggctgatg aacctggcta cgcacaaatc aataccgaag acgccaaacg tctgggtatt
1861 gaagatgagg cattggtttg ggtgcactcg cgtaaaggca aaattatcac ccgtgcgcag
1921 gtcagcgatc gtccgaacaa aggggcgatt tacatgacct accagtggtg gattggtgcc
1981 tgtaacgagc tggttaccga aaacttaagc ccgattacga aaacgccgga atacaaatac
2041 tgcgccgtgc gcgtcgagcc gatcgccgat cagcgcgccg ccgagcagta cgtgattgac
2101 gagtacaaca agttgaaaac tcgcctgcgc gaagcggcac tggcg

Claims (4)

1. produce hydrogen functional gene carrier pET32a-fdhF-1, which is characterized in that the genophore is includes Escherichia coli first The pET32a plasmids of dehydrogenase gene fdhF.
2. production hydrogen functional gene carrier pET32a-fdhF-1 according to claim 1, it is characterised in that the Escherichia coli The DE3 bacterial strains of Escherichia coli BL21.
3. the method for structure production hydrogen functional gene carrier pET32a-fdhF-1 as claimed in claim 1 or 2, which is characterized in that Include the following steps：
(1) primer of design E. coli formate dehydrogenase gene fdhF, and PCR amplification E. coli formate dehydrogenase gene fdhF；
(2) genetic fragment of recycling formate dehydrogenase gene fdhF carries out the integrality of sequence verification amplification gene；
(3) using I double digestion pET32a carriers of restriction enzyme Nde I and Xho, gel extraction obtains linearized vector；From It is connected on the cloning vector of fdhF genetic fragments, digestion simultaneously recycles fdhF segments；
(4) ready linearisation pET32a carriers and fdhF segments are connected by T4DNA ligases, obtained containing gene The recombinant vector of fdhF；
(5) recombinant vector is converted into DH5 α competent bacterias, obtains the pET32a-fdhF-1 carriers correctly built.
4. there is the application of the carrier pET32a-fdhF-1 of production hydrogen functional gene in Escherichia coli, which is characterized in that will by right The 1 or 2 production hydrogen functional gene carrier pET32a-fdhF-1 is asked to be transformed into Escherichia coli, this is cultivated in hydrogen culture medium is produced Escherichia coli produce hydrogen.
CN201810093583.5A 2018-01-31 2018-01-31 There is the carrier pET32a-fdhF-1 for producing hydrogen functional gene and its structure and application in Escherichia coli Pending CN108220319A (en)

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