CN109402033A - A kind of efficient hydrogenlike silicon ion engineering bacteria and its construction method using glucose - Google Patents
A kind of efficient hydrogenlike silicon ion engineering bacteria and its construction method using glucose Download PDFInfo
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- CN109402033A CN109402033A CN201811233149.9A CN201811233149A CN109402033A CN 109402033 A CN109402033 A CN 109402033A CN 201811233149 A CN201811233149 A CN 201811233149A CN 109402033 A CN109402033 A CN 109402033A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
Abstract
The present invention relates to a kind of hydrogenlike silicon ion engineering bacterias and its construction method for efficiently utilizing glucose, it for the hydrogenlike silicon ion 2.4.1 engineered strain for knocking out fruA, gcd gene, is obtained by knocking out fruA, gcd gene with carrier pK18mobsacB::fruA-LR, pK18mobsacB::gcd-LR for being connected with fruA-LR, gcd-LR sequence；The glucose of the hydrogenlike silicon ion bacterial strain consumption unit mass reduces production cost, shows good application prospect beneficial to obtaining more somatic cells.
Description
Technical field
The present invention relates to hydrogenlike silicon ion production field, especially a kind of hydrogenlike silicon ion engineering for efficiently utilizing glucose
Bacterium and its construction method.
Background technique
Hydrogenlike silicon ion is rich in a variety of nutrition medicinal ingredients, has anti peroxidation of lipid, anti-radiation, antitumor, adjusting machine
The functions such as body is immune, have new opplication in field of food and medicine.Ubiquinone can be extracted from thallus10, lycopene, vitamin
B12, the nutrition medicinal ingredient such as superoxide dismutase.Currently with hydrogenlike silicon ion fermented coenzyme Q10Industrialization is realized.With
Research go deep into, in field of food and medicine, more potential function will be mined out the bacterium, obtain a large amount of somatic cells
It is of great significance to above-mentioned nutrition medicinal ingredient is produced.
Glucose is a kind of most wide and mostly important monosaccharide of distributed in nature, it is a kind of polyhydroxy aldehyde.In biology
Field has critical role, be living cells energy source and metabolic intermediate product, i.e. biological main Energy supply material, because
This efficiently grows it using glucose and is of great significance in microorganism growth process.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of hydrogenlike silicon ion engineering bacteria for efficiently utilizing glucose.
Another technical problem to be solved by this invention is to provide the above-mentioned efficient hydrogenlike silicon ion work using glucose
The construction method of journey bacterium.
In order to solve the above technical problems, the technical scheme is that
A kind of efficient hydrogenlike silicon ion engineering bacteria using glucose, for the hydrogenlike silicon ion for knocking out fruA, gcd gene
(hydrogenlike silicon ion (Rhodobacter sphaeroides) 2.4.1 engineered strain.
Preferably, the above-mentioned efficient hydrogenlike silicon ion engineering bacteria using glucose, be by be connected with fruA-LR,
Carrier pK18mobsacB::fruA-LR, pK18mobsacB::gcd-LR of gcd-LR sequence knock out fruA, gcd gene and obtain
's.The method achieve the seamless knockout of specificity, and it is high-efficient, stablize, it is repeatable to obtain identical hydrogenlike silicon ion work
Journey bacterium.
The above-mentioned efficient hydrogenlike silicon ion engineering bacteria specificity using glucose is seamless to knock out the specific of fruA, gcd gene
Construction method: host strain used is hydrogenlike silicon ion (Rhodobacter sphaeroides) 2.4.1, and the carrier is
pK18mobsacB::fruA-LR,pK18mobsacB::gcd-LR；Knockout carrier pK18mobsacB::fruA-LR,
PK18mobsacB::gcd-LRization is gone in large intestine S17-1 competence, then by the method for engagement transfer, will be carried containing knocking out
The S17-1 and hydrogenlike silicon ion (Rhodobacter sphaeroides) 2.4.1 co-incubation of body, knockout carrier
PK18mobsacB::fruA-LR, pK18mobsacB::gcd-LR will be fed to hydrogenlike silicon ion (Rhodobacter
Sphaeroides) 2.4.1 then occurs left and right arms Homo~logous exchange and obtains knock-out bacterial strain, and homologous recombination principle is shown in Fig. 8, extracts
Genome is verified, if fruA gene knockout, is verified by PCR, it will the segment of 2015bp size is amplified, if
Gcd gene knockout, is verified by PCR, it will amplifies the segment of 1815bp size, i.e. acquisition fruA and gcd Gene Double knocks out
Hydrogenlike silicon ion bacterial strain.
Preferably, the above-mentioned efficient hydrogenlike silicon ion engineering bacteria using glucose, the carrier pK18mobsacB::
It include fruA left arm right arm 1819bp overlap fruA-LR in fruA-LR, wherein fruA left arm 979bp in hydrogenlike silicon ion
Sequence fruA-L, gene order are shown in sequence table 1,2；FruA right arm 834bp sequence fruA-R, gene order see sequence table 3,
4。
Preferably, the above-mentioned efficient hydrogenlike silicon ion engineering bacteria using glucose, the fruA left arm gene fruA-L and
FruA right arm gene fruA-R obtains overlapping genes fruA-LR by over-lap PCR, by overlapping genes fruA-LR and the carrier that sets out
PK18mobsacB with being attached after EcoRI and SalI double digestion, constructs the knockout carrier of fruA-LR gene, is respectively
PK18mobsacB::fruA-LR, plasmid map are shown in that Fig. 2, PCR verification result are shown in that Fig. 3, double digestion verification result are shown in Fig. 4.
Preferably, the above-mentioned efficient hydrogenlike silicon ion engineering bacteria using glucose, the carrier pK18mobsacB::gcd-
It include gcd left arm right arm 1709bp overlap gcd-LR in LR, wherein gcd left arm 770bp gene gcd- in hydrogenlike silicon ion
L, gene order are shown in sequence table 5,6；Gcd right arm 976bp gene gcd-R, gene order are shown in sequence table 7,8.
Preferably, the above-mentioned efficient hydrogenlike silicon ion engineering bacteria using glucose, gcd left arm the gene gcd-L and gcd
Right arm gene gcd-R obtains overlapping genes gcd-LR by over-lap PCR, by overlapping genes gcd-LR and the carrier that sets out
PK18mobsacB with being attached after EcoRI and SalI double digestion, constructs the knockout carrier of gcd-LR gene, is respectively
PK18mobsacB::gcd-LR, plasmid map are shown in that Fig. 5, PCR verification result are shown in that Fig. 6, double digestion verification result are shown in Fig. 7.
The hydrogenlike silicon ion bacterial strain that above-mentioned acquisition fruA and gcd Gene Double knock out is surveyed compared with wild-type strain otherness
Fixed its growth curve, glucose consumption rate etc..
Culture medium needed for the above-mentioned efficient hydrogenlike silicon ion engineering bacteria using glucose is as follows:
NH1 fluid nutrient medium: 8g/L yeast extract, 3g/L glucose, 2g/L NaCl, 1.3g/L KH2PO4,
0.125g/L MgSO4,1mg/L thiamine hydrochloride, 15 μ g/L biotins, 1mg/L niacin adjust pH to 7.2 with NaOH, and 115 DEG C go out
Bacterium 20 minutes.
1.5% agar is added in solid medium.
NH1+kana fluid nutrient medium: on the basis of NH1 fluid nutrient medium, kanamycins is added, kanamycins is final concentration of
25μg/mL。
SMM culture medium: glucose 8g/L, K2HPO43.5g/L, (NH4)2SO40.5g/L, NaCl 0.5g/L, KOH 2g/
L, CB mother liquor 20mL/L, growth factor mother liquor 1mL/L adjust pH to 6.9.
CB mother liquor: being added l0g nitrilotriacetic acid and 8g KOH in the deionized water of 500ml, successively adds after all dissolutions
Enter 14.6g anhydrous magnesium sulfate, 1.7gCaCl2·2H2O, 50mL M44 mother liquor.
M44 mother liquor: 2g/LEDTA, 11g/LZnS7H2O, 5g/LFeSO47H2O, 1.5g/LMnSO4H2O,
0.4g/L CuSO45H2O, 0.12/Lg H3BO3,0.37g/LCoCl46H2O.
Growth factor mother liquor: 4mg biotin, 200mg niacin, 100mg thiamine hydrochloride are added in 100ml deionized water
Element is settled to 200ml, filtration sterilization.
SMM+10%sucrose solid medium: the sucrose of addition l00g/L in SMM culture medium, the agar of 15g/L,
115 DEG C of sterilizing 20min.
Fermentation condition needed for the above-mentioned efficient hydrogenlike silicon ion engineering bacteria using glucose: 100mL fermentation liquid, inoculum concentration
2%, 30 DEG C, pH 6.9, revolving speed 200rpm.
Above-mentioned culture medium can be used standard method and prepare.
The beneficial effects of the present invention are:
The present invention changes bacterial strain glucose metabolic pathways by genetic modification, obtains one plant of class that can efficiently utilize glucose
Ball Rhodobacter strain compares wild-type strain, consumes the glucose of unit mass beneficial to obtaining more somatic cells, reduces production
Cost shows good application prospect.Specifically,
The present invention utilizes homologous recombination technique by the glucose PTS transport protein IIBC and pericentral siphon grape in hydrogenlike silicon ion
Glucocorticoid dehydrogenase encoding gene carries out seamless knockout, obtains the efficient red thin using glucose ball of fruA and gcd Gene Double knockout
Bacterium engineering bacteria recombinant bacterium.The recombinant bacterium that the present invention knocks out fruA, gcd Gene Double is trained by sole carbon source chemical synthesis of glucose
It supports base and carries out Analysis offermehtations, detect cell concentration and concentration of glucose in fermentation liquid, discovery maximum biomass is approximately wild strain
1.3 times, unit dry cell weight reaches 0.62g/g to glucose yield after fruA and gcd are knocked out, and improves 31% than wild strain.
The present invention significantly improves hydrogenlike silicon ion dry cell weight to glucose yield, and beneficial to obtaining more somatic cells, reduction is produced into
This.Wherein,
1, the present invention obtains the gene knock-out bacterial strain of fruA, gcd using homologous recombination technique, does not have to insertion selection markers,
Seamless fixed point knocks out fruA, gcd gene, does not influence the transcriptional level of its upstream and downstream gene.
2, the engineering bacteria for knocking out fruA, gcd gene is carried out Analysis offermehtations by the present invention, and discovery maximum biomass is approximately wild
1.3 times of strain, unit dry cell weight reaches 0.62g/g to glucose yield, improves 31% than wild strain.
3, the present invention efficiently will pass through microbial fermentation using the hydrogenlike silicon ion engineered strain of glucose, and analysis is found,
Dry cell weight significantly improves glucose yield, beneficial to obtaining more somatic cells, reduces production cost.
Detailed description of the invention
Fig. 1 is the building map of carrier pK18mobsacB of the present invention；
Fig. 2 is the building map of knockout carrier pK18mobsacB::fruA-LR of the present invention；
Fig. 3 is the PCR proof diagram of present invention building knockout carrier pK18mobsacB::fruA-LR, M:DNA Marker；
N: negative control；P: using fruA-LR segment as the positive control of template, amplification obtains fruA-LR segment, size 1819bp；
Segment is successfully connected on carrier, and amplification obtains fruA-LR segment, and size 1819bp is identical as positive control；
Segment fruA-LR is carried out double digestion with EcoRI and SalI for the present invention by Fig. 4, is processed with through same restriction endonuclease
Plasmid fragments pK18mobsacB be attached, bacterium colony PCR verifying and digestion verification, obtain knockout carrier pK18mobsacB::
fruA-LR。
Fig. 5 is the building map of knockout carrier pK18mobsacB::gcd-LR of the present invention；
Fig. 6 is the PCR proof diagram of present invention building knockout carrier pK18mobsacB::gcd-LR, M:DNA Marker；N:
Negative control；P: using gcd-LR segment as the positive control of template, amplification obtains gcd-LR segment, size 1709bp；Segment
The bacterial strain being successfully connected on carrier, amplification obtain gcd-LR segment, and size 1709bp is identical as positive control；
Fig. 7 is that segment gcd-LR is carried out double digestion with EcoRI and SalI by the present invention, and processed through same restriction endonuclease
Plasmid fragments pK18mobsacB is attached, and bacterium colony PCR verifying and digestion verification obtain knockout carrier pK18mobsacB::
gcd-LR。
Fig. 8 is the homologous recombination schematic diagram of fruA, gcd knock-out bacterial strain of the present invention screening；
Fig. 9 is fruA gene knock-out bacterial strain PCR proof diagram of the present invention, M:DNA Marker；N: negative control；P: wild
Type；1~2: knocking out the bacterial strain of fruA gene；
Figure 10 is gcd gene knock-out bacterial strain PCR proof diagram of the present invention, M:DNA Marker；N: negative control；P: wild
Type；1~3: knocking out the bacterial strain of gcd gene；
Figure 11 is present invention measurement wild type and the bis- growth curve maps after striking strain fermentation processing of fruA, gcd.
Specific embodiment
In order to make those skilled in the art better understand technical solution of the present invention, With reference to embodiment
Technical solution of the present invention is described in further detail.
The present invention utilizes homologous recombination technique by the glucose PTS transport protein IIBC and pericentral siphon grape in hydrogenlike silicon ion
Glucocorticoid dehydrogenase encoding gene fruA and gcd carry out seamless knockout, obtain glucose PTS transport protein IIBC and pericentral siphon glucose is de-
The engineered strain of hydrogenase gene afunction, the contents of the present invention include: the method for constructing fruA the and gcd knockout carrier,
The method for constructing fruA and gcd gene knock-out bacterial strain.The carrier is pK18mobsacB, includes sacB gene in carrier.
FruA-L, fruA-R of the present invention derive from hydrogenlike silicon ion (Rhodobacter sphaeroides)
2.4.1, fruA-LR is the sequence that fruA-L, fruA-R link together, and is to pass through overlapping by template of fruA-L, fruA-R
What round pcr expanded；Described gcd-L, the gcd-R derives from hydrogenlike silicon ion (Rhodobacter sphaeroides)
2.4.1, gcd-LR is the sequence that gcd-L, gcd-R link together, and is to pass through over-lap PCR skill by template of gcd-L, gcd-R
What art expanded.
Described knockout carrier pK18mobsacB::fruA-LR, pK18mobsacB::gcd-LR be respectively by fruA-LR,
Gcd-LR segment is inserted into starting vector pK18mobsacB, construct knockout carrier pK18mobsacB::fruA-LR,
PK18mobsacB::gcd-LR, engagement are transferred in hydrogenlike silicon ion, obtain fruA and gcd gene knock-out bacterial strain.Above-mentioned bacterial strains
After fermented culture, the fermentation liquid of different incubation times is taken to carry out the detection of growth curve.
Bacillus subtilis sacB gene encodes secreting type sucrose levanase, which can be catalyzed sucrose hydrolysis and synthesis is high
The levulan of molecular weight.There are potential toxic effects to cell for the accumulation of high molecular weight levulan, can cause cell death.Also
It is to say, sacB gene makes the characteristic of bacterium acquisition sucrose sensitivity.This research is it has been found that the sucrose 5% or 5% or more exists
Under, the expression of sacB gene can cause hydrogenlike silicon ion (Rhodobacter sphaeroides) 2.4.1 dead.Based on this
Kind characteristic, sacB gene are genetically applied to the gene functional research of hydrogenlike silicon ion as a kind of reversed selection markers
On, the lethal effect in the presence of 10% sucrose (in the presence of 10% sucrose, bacterium is all dead).Concrete operation step of the present invention is such as
Under:
One, the building of knockout carrier pK18mobsacB::fruA-LR, pK18mobsacB::gcd-LR
1, with the genome of hydrogenlike silicon ion (Rhodobacter sphaeroides) 2.4.1 (from Zhejiang University)
FruA-L is obtained by PCR amplification using primer fruAL-F, fruAL-R and fruAR-F, the fruAR-R in table 1 for template
And fruA-R；Using primer gcdL-F, gcdL-R and gcdR-F, the gcdR-R in table 2, by PCR amplification obtain gcd-L and
gcd-R。
Specific PCR reaction system are as follows:
Specific PCR reaction condition are as follows:
Complete target fragment fruA-L, fruA-R and gcd-L, gcd-R are obtained after PCR.
2, using fruA-L, fruA-R and gcd-L, gcd-R as template, be utilized respectively primer fruAL-F in table 1,
Primer gcdL-F, gcdR-R in fruAR-R and table 2 carry out over-lap PCR amplification and obtain fruA-LR and gcd-LR.
Specific PCR reaction system are as follows:
Specific PCR reaction condition are as follows:
Complete target fragment fruA-LR and gcd-LR is obtained after PCR.
1 the primer sequence of table
2 the primer sequence of table
By acquired DNA fragmentation fruA-LR, gcd-LR with EcoRI (be purchased from TaKaRa company, article No. D1040A) and
SalI (being purchased from TaKaRa company, article No. D1080A) carries out double digestion, after recycling and through the processed plasmid piece of same restriction endonuclease
Section pK18mobsacB is attached, and connection product is converted to escherichia coli jm109 competent cell, and be spread evenly across band
On the LB plate for having kanamycins (100 μ g/ml), 37 DEG C of overnight incubations, picking monoclonal carries out bacterium colony PCR verifying and digestion
Verifying obtains knockout carrier pK18mobsacB::fruA-LR, pK18mobsacB::gcd-LR, proof diagram such as Fig. 4 and Fig. 7.
Two, the building of fruA, gcd gene knock-out bacterial strain
Above-mentioned acquired knockout carrier engagement is transferred to hydrogenlike silicon ion, method particularly includes:
(1) three rides are on NH1 solid plate from the hydrogenlike silicon ion glycerol tube of -80 DEG C of preservations, and in 30 DEG C of perseverances
It is clear to single colonie that culture is inverted in warm incubator；
(2) after 100 μ l recovery in the supported competent cell for being transferred to Escherichia coli S17-1 built, being drawn
Bacterium solution is coated on the solid plate of LB+kana, and culture left and right for 24 hours is inverted in 37 DEG C of constant incubators；
(3) hydrogenlike silicon ion and Escherichia coli S17-1 are picked them separately into monoclonal and is inoculated in NH1 fluid nutrient medium and LB liquid
In body culture medium (adding the antibiotic of respective concentration according to carrier resistance), hydrogenlike silicon ion is trained in 30 DEG C, 200rpm shaking table
Left and right, Escherichia coli S17-1 for 24 hours is supported to be incubated overnight in 37 DEG C, 200rpm shaking table；
(4) it respectively takes 1mL bacterium solution in 1.5mL EP pipe, after 8000rpm is centrifuged 3min, discards supernatant；
(5) primary with sterile water washing thalline precipitating；
(6) bacterial sediment is resuspended in the fluid nutrient medium for being separately added into proper amount of fresh；
(7) two kinds of bacterium solutions are mixed in different ratios, draws mixed bacteria liquid point in plate in sterilised membrane filter with pipette tips
Centre, dries up bacterium solution in superclean bench；
(8) plate is just being placed in 30 DEG C of constant incubators after bacterium solution drying and is cultivating left and right for 24 hours；
(9) lawn on plate is washed down with 400 μ l sterile waters, is coated on NH1+kana+K2TeO3(kana concentration is
25mg/L, K2TeO3Concentration is 150mg/L) solid plate on, the stationary culture in 30 DEG C of constant incubators；
(10) to there is black transformant to grow on plate, picking monoclonal is inoculated in NH1+kana fluid nutrient medium and in 32
DEG C, left and right for 24 hours is cultivated in 200rpm temperature control shaking table
(11) it finally crosses and is isolated and purified on NH1+kana solid plate
Three, the screening of fruA, gcd knock-out bacterial strain, the specific method is as follows:
(1) picking monoclonal is inoculated in NH1+kana fluid nutrient medium, is cultivated in 30 DEG C, 200rpm temperature control shaking table
Left and right for 24 hours；
(2) it extracts genomic DNA and carries out PCR amplification, whether verifying single exchange strains are correct；
(3) monoclonal that picking verifies correct single exchange strains, which is inoculated in NH1 fluid nutrient medium, (does not add any antibiosis
Element), left and right for 24 hours is cultivated in 30 DEG C, 200rpm temperature control shaking table；
(4) it transfers by 1% inoculum concentration, and bacterium solution is diluted to suitable concentration, draw 100 μ l and be uniformly coated with
In on the solid plate of SMM+10%sucrose, double crossing over bacterial strain is screened；
(5) bacterium solution after transferring cultivates left and right for 24 hours in 30 DEG C, 200rpm temperature control shaking table, and repeats to walk experimental implementation；
(6) it is grown to transformant on SMM+10%sucrose solid plate, picking single colonie is inoculated in NH1 Liquid Culture
Left and right for 24 hours is cultivated in base in 30 DEG C, 200rpm temperature control shaking table；
(7) it extracts genomic DNA and carries out PCR amplification, whether verifying double crossing over bacterial strain is correct.Utilize drawing in table 3, table 4
The bacterial strain of object P1, P4 verifying generation homologous recombination, homologous recombination schematic diagram such as Fig. 6, using the genome of bacterial strain to be detected as template,
If there is no homologous recombination, with primer P1, P4 amplify come clip size be respectively 3736bp, 3229bp；If
Homologous recombination has occurred, then clip size is respectively 2015bp, 1815bp, such as Fig. 9, Figure 10.
3 the primer sequence of table
4 the primer sequence of table
Four, fruA, gcd Gene Double knock-out bacterial strain growth detection
FruA, gcd gene knock-out bacterial strain ferment in SMM liquid culture medium as described above.
Fermentation condition: 100mL fermentation liquid, inoculum concentration 2%, 30 DEG C, pH 6.9, revolving speed 200rpm, ferment 120h or more, often
12h takes one time fermentation liquid.Testing result such as the following table 5 and Figure 11.
5 wild-type strain of table and the bis- knock-out bacterial strain growth datas of fruA, gcd
It is above-mentioned that the hydrogenlike silicon ion engineering bacteria of glucose and its building are efficiently utilized to the one kind referring to specific embodiment
The detailed description that method carries out, is illustrative without being restrictive, can enumerate several implementations according to limited range
Example, therefore the change and modification in the case where not departing from present general inventive concept, should belong within protection scope of the present invention.
Sequence table
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Claims (7)
1. a kind of hydrogenlike silicon ion engineering bacteria for efficiently utilizing glucose, it is characterised in that: for the class for knocking out fruA, gcd gene
The red bacterium 2.4.1 engineered strain of ball.
2. the efficient hydrogenlike silicon ion engineering bacteria using glucose according to claim 1, it is characterised in that: be to pass through use
Carrier pK18mobsacB::fruA-LR, the pK18mobsacB::gcd-LR for being connected with fruA-LR, gcd-LR sequence are knocked out
What fruA, gcd gene obtained.
3. the efficient hydrogenlike silicon ion engineering bacteria using glucose according to claim 2, it is characterised in that: efficiently utilize
The seamless specific construction method for knocking out fruA, gcd gene of hydrogenlike silicon ion engineering bacteria specificity of glucose are as follows: host strain used
For hydrogenlike silicon ion (Rhodobacter sphaeroides) 2.4.1, the carrier be pK18mobsacB::fruA-LR,
pK18mobsacB::gcd-LR；Knockout carrier pK18mobsacB::fruA-LR, pK18mobsacB::gcd-LRization are gone to greatly
In intestines S17-1 competence, then by the method for engagement transfer, the S17-1 containing knockout carrier and hydrogenlike silicon ion 2.4.1 are total to
With culture, knockout carrier pK18mobsacB::fruA-LR, pK18mobsacB::gcd-LR will be fed to hydrogenlike silicon ion
2.4.1, then, verified by extracting genome, if fruA gene knockout, is verified by PCR, it will amplify
The segment of 2015bp size, if gcd gene knockout, is verified by PCR, it will the segment for amplifying 1815bp size obtains
Obtain the hydrogenlike silicon ion bacterial strain that fruA and gcd Gene Double knocks out.
4. the efficient hydrogenlike silicon ion engineering bacteria using glucose according to claim 2, it is characterised in that: the carrier
It include fruA left arm right arm 1819bp overlap fruA-LR in pK18mobsacB::fruA-LR, wherein in hydrogenlike silicon ion
FruA left arm 979bp sequence fruA-L, gene order are shown in sequence table 1,2；FruA right arm 834bp sequence fruA-R, gene
Sequence is shown in sequence table 3,4.
5. the efficient hydrogenlike silicon ion engineering bacteria using glucose according to claim 4, it is characterised in that: the fruA
Left arm gene fruA-L and fruA right arm gene fruA-R obtains overlapping genes fruA-LR by over-lap PCR, by overlapping genes
FruA-LR and the carrier pK18mobsacB that sets out construct fruA-LR gene respectively with being attached after EcoRI and SalI double digestion
Knockout carrier, be pK18mobsacB::fruA-LR.
6. the efficient hydrogenlike silicon ion engineering bacteria using glucose according to claim 2, it is characterised in that: the carrier
It include gcd left arm right arm 1709bp overlap gcd-LR in pK18mobsacB::gcd-LR, wherein gcd in hydrogenlike silicon ion
Left arm 770bp gene gcd-L, gene order are shown in sequence table 5,6；Gcd right arm 976bp gene gcd-R, gene order are shown in sequence
List 7,8.
7. the efficient hydrogenlike silicon ion engineering bacteria using glucose according to claim 6, it is characterised in that: the gcd
Left arm gene gcd-L and gcd right arm gene gcd-R obtains overlapping genes gcd-LR by over-lap PCR, by overlapping genes gcd-LR
With the carrier pK18mobsacB that sets out respectively with being attached after EcoRI and SalI double digestion, the knockout of building gcd-LR gene is carried
Body is pK18mobsacB::gcd-LR.
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| CN2017110379573 | 2017-10-31 | ||
| CN201711037957 | 2017-10-31 |
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