CN108795969A - One plant of engineering bacteria NXdP and its construction method and application - Google Patents
One plant of engineering bacteria NXdP and its construction method and application Download PDFInfo
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Abstract
The present invention provides one plant of engineering bacteria NXdP and its construction method and applications, belong to genetic engineering and technical field of biological materials.The deposit number of engineering bacteria NXdP of the present invention is CGMCC15406, and preservation place is China General Microbiological culture presevation administrative center, and the preservation time is on 03 01st, 2018.The engineering bacteria NXdP obtained using present invention structure only generates hydrogel (a surname's good fortune glue) in fermentation, finally obtained sterling yield is 15~22g/L, total output is 30~35g/L, conversion ratio is 48% or more, the yield of hydrogel can be effectively improved, and there is very high carbohydrate gum conversion ratio, which has extensive prospects for commercial application.
Description
Technical field
The invention belongs to genetic engineerings and technical field of biological materials, and in particular to one plant of engineering bacteria NXdP and its structure side
Method and application.
Background technology
Sphingomonas (Sphingomonas sp.) is to breathe quinone type and cell polar according to 16s rRNA sequences
Property one of the feature extractions such as fat pattern new belong to.The important feature for being different from other Gram-negative bacterias is contained in its cell membrane
Glycosyl sphingolipid, and without lipopolysaccharides.Sphingomonas is distributed widely in water body, in soil and air.Currently, for sphingol list
The research of born of the same parents Pseudomonas is concentrated mainly on the ability that the Pseudomonas has degradation persistent organic pollutants, for example dibenzofuran can quilt
Sphingomonas sp.RW1 are utilized as unique carbon source material；Ability with synthesis bata-carotene；It is a kind of with synthesis
The ability of acid capsular polysaccharide, the structure of these capsular polysaccharides is similar but is not quite similar, and is referred to as sphingol glue.
Currently, mass production and widely applied sphingol glue includes mainly：Sphingomonas elodea
The gellan gum of ATCC3161 synthesis, the welan gum of Sphingomonas sp.ATCC31555 synthesis, Sphingomonas
Enlightening spy's glue of sp.ATCC53159 synthesis, the sandlwood glue etc. of Sphingomonas sp.ATCC31961 synthesis.This quasi-sheath ammonia alcohol glue
With relatively conservative backbone structure, and the type of side-chain radical, position then have great diversity, this makes the knot of sphingol
Structure and function are more abundant, to impart each unique physical property of sphingol glue.Such as the not no knot of sugar side chains
Cold glue can form gel, to be widely used in food, daily use chemicals and medically；With rhamnose or sweet dew carbohydrate side chain
Welan gum, acidproof, alkaline-resisting, heat safe highly viscous solution can be formed, with one or two sandlwood carbohydrate side chain Di Te
Glue can form highly viscous solution under low consistency conditions, be widely used in high-tech sectors such as building, drilling well, oil recoveries；With
The development of biotechnology, the bacterial strain that can more and more produce sphingol glue is identified, and these newfound natural polymers
Resource will be played in following biogum application and more importantly be acted on.
Sphingol single-cell Sphingomonas sp.T-3 (CGMCC No.10150), which are one plant, can produce transparent aquagel
Production bacterial strain (Sphingol single-cell T-3 and its common fermentation production biological polyoses and poly- β hydroxybutyric acids method, authorized
CN201510110078.3).But a large amount of this by-products of accumulating poly β hydroxybutyric acids in the cell, affect the production of hydrogel
Amount and purity, in the art, there is no a kind of engineering bacterias, it is possible to reduce or even do not generate poly- β hydroxybutyric acids completely.
Invention content
In view of this, the purpose of the present invention is to provide a kind of engineering bacteria NXdP of production hydrogel, poly- β hydroxyls fourth is not generated
Acid realizes the high conversion of carbohydrate gum.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
It is described the present invention provides the engineering bacteria NXdP (Sphingomonas sanxanigenens) of one plant of production hydrogel
The deposit number of engineering bacteria NXdP is CGMCC15406, and preservation place is China General Microbiological culture presevation administrative center, tool
Body address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, and the preservation time is 2018 03
The moon 01.
The present invention provides a kind of methods of the above-mentioned engineering bacteria NXdP of structure, include the following steps：
1) double crossing over methods of homologous recombination is utilized to knock out the phbB bases of Sphingol single-cell (Sphingomonas sp.T-3)
Cause obtains the engineering bacteria T-3- Δs PHB for not producing poly- β hydroxybutyric acids；
2) the engineering bacteria T-3- Δs PHB that step 1) obtains is cultivated in TPG fluid nutrient mediums, carried out after culture etc.
Ion mutagenesis obtains mutagenesis engineering bacteria T-3- Δs PHB；The TPG fluid nutrient mediums include：8~12g/L of glucose, peptone 3~
1~5g/L of 7g/L, 1~5g/L of yeast powder and beef extract powder；
3) the mutagenesis engineering bacteria T-3- Δs PHB that step 2) obtains is cultivated on TPG solid mediums, screens water-setting
The highest bacterial strain of glue yield, the hydrogel yield highest bacterial strain are the engineering bacteria NXdP for producing hydrogel；The TPG solid cultures
Base includes：8~12g/L of glucose, 3~7g/L of peptone, 1~5g/L of yeast powder, 1~5g/L of beef extract powder and agar powder 15g/
L。
Preferably, step 1) the double crossing over methods of homologous recombination, includes the following steps：
A) using Sphingol single-cell T-3 genomic DNAs template, SEQ ID NO are utilized：The primer of sequence shown in 1~4 into
Recombinant fragment is recycled to obtain in row over-lap PCR, purifying；
B) recombinant fragment and pLO3 described in restriction enzyme SacI and XbaI or SacI and PacI difference double digestions are utilized
Digestion products are attached by plasmid, obtain recombinant plasmid pLO3- Δs gene；
C) the recombinant plasmid pLO3- Δs gene that step b) is obtained is transformed into E.Coli S17 competent cells,
Obtain E.Coli S17/pLO3- Δs gene；
D) single bacterium colony of the obtained E.Coli S17/pLO3- Δs gene of step c) is being contained into tetrLB liquid
6~10h is cultivated in culture medium, and E.Coli S17/pLO3- Δ gene thalline are collected by centrifugation；
E) Sphingol single-cell T-3 single bacterium colonies are selected to be inoculated into containing CmrSeed culture medium in, cultivate 20~28h, centrifugation
Collect Sphingol single-cell T-3 thalline；
F) by the E.Coli S17/pLO3- Δ gene thalline that step d) is obtained and the Sphingol single-cell that step e) is obtained
T-3 thalline use MgSO respectively4After solution is resuspended, according to 1：The volume ratio mixing of (1.5~2.5) filters, in TPG solid mediums
10~15h is shifted in upper engagement, obtains single-swap recon；
G) the single-swap recon that step f) is obtained is inoculated in the seed culture medium of non-resistant, is passed on twice,
F2 is obtained for single-swap recon；The seed culture medium includes：8~12g/L of sucrose, 1.8~3g/L of peptone, yeast powder 1~
2.5g/L, K2HPO41.8~3g/L and MgSO40.06~0.15g/L；
H) F2 that step g) is obtained is seeded to for single-swap recon on the TPG solid mediums containing sucrose, is trained
65~78h is supported, double exchange reorganization of phbB gene lists copy must be knocked out；
I) double exchange reorganization of the knockout phbB gene lists copy obtained using step h) utilizes SEQ ID as template
The primer of sequence shown in NO.9~12 repeats step a)~h), double exchange reorganization of phbB Gene Doubles copy must be knocked out；
J) using it is described knockout phbB Gene Doubles copy double exchange reorganization as template, successively utilize SEQ ID NO.17~
20, the primer of NO.23~26 SEQ ID, NO.27~30 SEQ ID and NO.33~36 SEQ ID repeats step a)~h), it obtains
The engineering bacteria T-3- Δs PHB of poly- β hydroxybutyric acids is not produced.
Preferably, the program of the step a) over-lap PCRs, including：1st~10 cycle, 98 DEG C of denaturation 10s, in 15s
55 DEG C are warming up to 72 DEG C of annealing, 72 DEG C of extension 1min；11st~20 cycle, 98 DEG C of denaturation 10s, is cooled to for 72 DEG C in 15s
55 DEG C of annealing, 72 DEG C of extension 2min；21st~31 cycle, 98 DEG C of denaturation 10s, 60 DEG C of annealing 15s, 72 DEG C of extension 2min；The
32 cycles, 72 DEG C of extension 10min.
Preferably, mass concentration of the step h) sucrose in TPG solid mediums is 8~12%.
The present invention also provides the engineering bacteria NXdP that a kind of engineering bacteria NXdP or the above method are built to produce hydrogel
In application.
Preferably, include the following steps：
1. the engineering bacteria NXdP single bacterium colonies are seeded in TPG fluid nutrient mediums, 20~26h of shake culture must be cultivated
Liquid；
2. the 1. culture solution that step obtains is seeded in seed culture medium, 20~26h of shake culture obtains seed
Liquid；
3. by step, 2. the seed liquor is inoculated in fermentation medium, and ferment 68~75h, obtains zymotic fluid；
4. the 3. zymotic fluid that step obtains is diluted with distilled water, heating centrifugation, isolated supernatant liquid and bacterium
Body precipitates；
5. the 4. supernatant liquid pH to 3.0 that regulating step obtains, collects the precipitation of generation, obtains hydrogel, be named as
Declare good fortune glue.
Preferably, the step 1. temperature with the step 2. shake culture, is independently 28~35 DEG C.
Preferably, the culture medium of the step 3. fermentation, including：30~70g/L of glucose, 0.5~2g/L of beancake powder,
K2HPO41~2g/L, MgSO40.1~1g/L and NaNO31~2g/L.
Preferably, step 3. the fermentation temperature be 28~35 DEG C.
The present invention provides one plant of engineering bacteria NXdP, the engineering bacteria NXdP provided by the invention not to generate poly- β hydroxyls fourth
Acid only generates a surname's good fortune glue, realizes the high conversion of carbohydrate gum, to improve the purity of hydrogel (a surname's good fortune glue).In conjunction with experiment
As a result with data it is found that when engineering bacteria NXdP of the present invention is applied to fermentation, sterling yield is 21.20 ± 0.38g/L, always
Yield is 32.03 ± 2.21g/L；Remaining glucose content is differed from 40g/L to 0g/L in different fermentations period zymotic fluid, is turned
Rate is 80%, and it is about 6.4pa.s that fermentation broth viscosity, which reaches maximum value, and obtained hydrogel (a surname's good fortune glue) range of shear rate
For 0.001s-1~1000s-1, the presence of poly- β hydroxybutyric acids is not detected.
Description of the drawings
Fig. 1 is the structure flow chart of knockout carrier；
Fig. 2 is the knockout and verification result figure that poly- β hydroxybutyric acids synthesize key gene；
Fig. 3 is NXdP strain fermentation Yield mappings；
Fig. 4 is the rheological property result figure for declaring good fortune glue.
Specific implementation mode
It is described the present invention provides the engineering bacteria NXdP (Sphingomonas sanxanigenens) of one plant of production hydrogel
The deposit number of engineering bacteria NXdP is CGMCC15406, and preservation place is China General Microbiological culture presevation administrative center, tool
Body address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, and the preservation time is 2018 03
The moon 01.
The present invention provides a kind of methods of the above-mentioned engineering bacteria NXdP of structure, include the following steps：
1) double crossing over methods of homologous recombination is utilized to knock out the phbB bases of Sphingol single-cell (Sphingomonas sp.T-3)
Cause obtains the engineering bacteria T-3- Δs PHB for not producing poly- β hydroxybutyric acids；
2) the engineering bacteria T-3- Δs PHB that step 1) obtains is cultivated in TPG fluid nutrient mediums, carried out after culture etc.
Ion mutagenesis obtains mutagenesis engineering bacteria T-3- Δs PHB；The TPG fluid nutrient mediums include：8~12g/L of glucose, peptone 3~
1~5g/L of 7g/L, 1~5g/L of yeast powder and beef extract powder；
3) the mutagenesis engineering bacteria T-3- Δs PHB that step 2) obtains is cultivated on TPG solid mediums, screens water-setting
The highest bacterial strain of glue yield, the highest bacterial strain of hydrogel yield are the engineering bacteria NXdP for producing hydrogel；The TPG solids training
Foster base includes：8~12g/L of glucose, 3~7g/L of peptone, 1~5g/L of yeast powder, 1~5g/L of beef extract powder and agar powder
15g/L。
The present invention knocks out Sphingol single-cell when building engineering bacteria NXdP, first with double crossing over methods of homologous recombination
The phbB genes of (Sphingomonas sp.T-3) obtain the engineering bacteria T-3- Δs PHB for not producing poly- β hydroxybutyric acids.It is of the present invention
Double crossing over methods of homologous recombination, preferably includes the following steps：
A) using Sphingol single-cell T-3 genomic DNAs template, SEQ ID NO are utilized：The primer of sequence shown in 1~4 into
Recombinant fragment is recycled to obtain in row over-lap PCR, purifying；
B) recombinant fragment and pLO3 matter described in restriction enzyme SacI and XbaI or acI and PacI difference double digestions are utilized
Grain, digestion products are attached, recombinant plasmid pLO3- Δs gene is obtained；
C) the recombinant plasmid pLO3- Δs gene that step b) is obtained is transformed into E.Coli S17 competent cells,
Obtain E.Coli S17/pLO3- Δs gene；
D) single bacterium colony of the obtained E.Coli S17/pLO3- Δs gene of step c) is being contained into tetrLB liquid
6~10h is cultivated in culture medium, and E.Coli S17/pLO3- Δ gene thalline are collected by centrifugation；
E) Sphingol single-cell T-3 single bacterium colonies are selected to be inoculated into containing CmrSeed culture medium in, cultivate 20~28h, centrifugation
Collect Sphingol single-cell T-3 thalline；
F) by the E.Coli S17/pLO3- Δ gene thalline that step d) is obtained and the Sphingol single-cell that step e) is obtained
T-3 thalline use MgSO respectively4After solution is resuspended, according to 1：The volume ratio mixing of (1.5~2.5) filters, in TPG solid mediums
10~15h is shifted in upper engagement, obtains single-swap recon；
G) the single-swap recon that step f) is obtained is inoculated in the seed culture medium of non-resistant, is passed on twice,
F2 is obtained for single-swap recon；The seed culture medium includes：8~12g/L of sucrose, 1.8~3g/L of peptone, yeast powder 1~
2.5g/L, K2HPO41.8~3g/L and MgSO40.06~0.15g/L；
H) F2 that step g) is obtained is seeded to for single-swap recon on the TPG solid mediums containing sucrose, is trained
65~78h is supported, double exchange reorganization of phbB gene lists copy must be knocked out；
I) double exchange reorganization of the knockout phbB gene lists copy obtained using step h) utilizes SEQ ID as template
The primer of sequence shown in NO.9~12 repeats step a)~h), double exchange reorganization of phbB Gene Doubles copy must be knocked out；
J) using double exchange reorganization of the knockout phbB Gene Doubles copy as template, such as SEQ ID NO.17 are utilized successively
~20, the primer of sequence shown in NO.23~26 SEQ ID, NO.27~30 SEQ ID and NO.33~36 SEQ ID repeats to walk
It is rapid a)~h), obtain the engineering bacteria T-3- Δs PHB for not producing poly- β hydroxybutyric acids.
The present invention utilizes SEQ ID NO using Sphingol single-cell T-3 genomic DNAs template：Sequence shown in 1~4 is drawn
Object carries out over-lap PCR, and recombinant fragment is recycled to obtain in purifying.In the present invention, the system of the over-lap PCR includes preferably template
1ul, each 0.4ul of upstream and downstream primer, the high-fidelity DNA polymerase Prime star 12.5ul of premix, add water polishing 25ul.This
The program for inventing the over-lap PCR includes preferably：1st~10 cycle, 98 DEG C of denaturation 10s, 72 are warming up to for 55 DEG C in 15s
DEG C annealing, 72 DEG C extension 1min；11st~20 cycle, 98 DEG C of denaturation 10s, 55 DEG C of annealing are cooled to for 72 DEG C in 15s, 72 DEG C
Extend 2min；21st~31 cycle, 98 DEG C of denaturation 10s, 60 DEG C of annealing 15s, 72 DEG C of extension 2min；32nd cycle, 72 DEG C
Extend 10min.
After obtaining recombinant fragment, the present invention distinguishes double digestion using restriction enzyme SacI and XbaI or SacI and PacI
The recombinant fragment and pLO3 plasmids, digestion products are attached, recombinant plasmid pLO3- Δs gene is obtained.It is of the present invention double
The temperature of digestion is preferably 32~40 DEG C, more preferably 35~38 DEG C, most preferably 37 DEG C.Double digestion of the present invention
Time is preferably 60~120min, more preferably 80~100min, most preferably 90min.The present invention is to double enzymes
There is no particular determinations for the system cut, and utilize the conventional double digestion method of this field.The present invention is excellent after the double digestion
Choosing further includes recovery purifying process, and there is no particular determinations for method and step of the present invention to the recovery purifying, utilize this
The conventional kit in field operates.
The enzyme of connection of the present invention includes preferably ligase, and the ligase is preferably T4 ligases.The present invention
The temperature of the connection is preferably 12~20 DEG C, more preferably 14~18 DEG C, most preferably 16 DEG C.Company of the present invention
The time connect is preferably 8~16h, more preferably 10~14h, most preferably 12h.System of the present invention to the connection
There is no particular determinations, utilize the conventional connection methods of this field.
After obtaining recombinant plasmid pLO3- Δs gene, recombinant plasmid pLO3- Δs gene is transformed into E.Coli S17 by the present invention
In competent cell, E.Coli S17/pLO3- Δs gene is obtained.When conversion of the present invention, recombinant plasmid pLO3- Δs gene with
The volume ratio of E.Coli S17 competent cells is preferably 1:10.Conversion of the present invention can be to recombinant plasmid pLO3- Δs
Gene is expanded.There is no particular determinations for method of the present invention to the conversion, are using the conventional transformation methods of this field
It can.
After obtaining E.Coli S17/pLO3- Δs gene, the present invention exists the single bacterium colony of E.Coli S17/pLO3- Δs gene
Contain tetrLB liquid medium in cultivate 6~10h, E.Coli S17/pLO3- Δ gene thalline are collected by centrifugation.The present invention
The amount of the single bacterium colony and LB liquid medium is 1/5mL, tet in the LB liquid mediumrMass concentration it is preferred
For 50~150 μ g/mL, more preferably 80~120 μ g/mL, most preferably 100 μ g/mL.Culture of the present invention is preferred
Temperature for shaking table culture, the culture is preferably 35~40 DEG C, more preferably 36~39 DEG C, most preferably 37 DEG C,
The rotating speed of the shaking table is preferably 180~220rpm, more preferably 190~210rpm, most preferably 200rpm.This hair
The time of the bright culture is preferably 7~9h, more preferably 8h.The rotating speed of centrifugation of the present invention is preferably 4000~
8000rpm, more preferably 5000~7000rpm, most preferably 6000rpm, time of the centrifugation is preferably 3~
8min, more preferably 4~6min, most preferably 5min.For the present invention after the centrifugation, obtained precipitation is E.Coli
S17/pLO3- Δ gene thalline.
The present invention selects Sphingol single-cell T-3 single bacterium colonies and is inoculated into containing CmrSeed culture medium in, cultivate 20~28h,
Sphingol single-cell T-3 thalline are collected by centrifugation.Seed culture medium of the present invention includes：8~12g/L of sucrose, peptone 1.8~
3g/L, yeast powder 1~2.5g/L, K2HPO41.8~3g/L and MgSO40.06~0.15g/L.Single bacterium colony of the present invention and kind
The amount of sub- culture medium is 1/5mL, Cm in the seed culture mediumrMass concentration be preferably 50~150 μ g/mL, it is more excellent
Choosing is 80~120 μ g/mL, most preferably 100 μ g/mL.Culture of the present invention is preferably shaking table culture, the culture
Temperature be preferably 25~35 DEG C, more preferably 28~32 DEG C, most preferably 30 DEG C, the rotating speed of the shaking table is preferred
For 180~220rpm, more preferably 190~210rpm, most preferably 200rpm.The time of culture of the present invention is preferred
It is 22~26h, more preferably 23~25h, most preferably for 24 hours.The rotating speed of centrifugation of the present invention is preferably 4000
~8000rpm, more preferably 5000~7000rpm, most preferably 6000rpm, time of the centrifugation is preferably 3~
8min, more preferably 4~6min, most preferably 5min.
After obtaining E.Coli S17/pLO3- Δ gene thalline and Sphingol single-cell T-3 thalline, the present invention uses respectively
MgSO4After solution is resuspended, according to 1：The volume ratio mixing of (1.5~2.5) filters, the engagement transfer 10 on TPG solid mediums
~15h obtains single-swap recon.Include preferably using MgSO before resuspension of the present invention4The process of solution washing, the washing
Use MgSO4The concentration of solution is preferably 8~12mmol/L, more preferably 9~11mmol/L, most preferably 10mmol/
L.The number of washing of the present invention is preferably 2 times, includes preferably gentle centrifugation process each time after washing.It is of the present invention
The rotating speed of gentle centrifugation is preferably 5000~7000rpm, and the time of gentle centrifugation is preferably 5~7min.Resuspension of the present invention
MgSO4Solution usage is preferably 150~250 μ L, more preferably 180~220 μ L, most preferably 200 μ L.The present invention
The volume ratio of the E.Coli S17/pLO3- Δ gene thalline and Sphingol single-cell T-3 thalline is preferably 1：(1.8~
2.2), more preferably 1:2.Suction filtration of the present invention is preferred to be filtered to filter membrane, and the aperture of the filter membrane is preferably
0.22μm.Filter membrane is preferably placed on TPG solid mediums by Conjugative tiansfer of the present invention, and the filter membrane preferably has thalline
On one side upward.The temperature of Conjugative tiansfer of the present invention is preferably 25~35 DEG C, more preferably 28~32 DEG C, most preferably
It is 30 DEG C.The time of Conjugative tiansfer of the present invention is preferably 11~13h, more preferably 12h.
After obtaining single-swap recon, the single-swap recon is inoculated in the seed culture medium of non-resistant by the present invention
In, passage twice, obtains F2 for single-swap recon.The present invention further includes preferably bacterium colony PCR verifications, institute before the inoculation
When stating bacterium colony PCR verifications, the MgSO of 200uL is preferably first used4Solution washs the thalline on filter membrane, is coated on containing CmrAnd tetr
Dual anti-solid TPG solid mediums on, 72h is cultivated in 30 DEG C of constant incubators, since pLO3 is in Sphingol single-cell T-3
In can not replicate, after pLO3- Δs gene engagement is transferred to T-3, can be exchanged by upstream homology arm or downstream homology arm whole
It is incorporated into genome.Picking single bacterium drops down onto 5ml containing in dual anti-seed fluid nutrient mediums of saccharomycete, in 30 DEG C of constant-temperature tables on dual anti-tablet
200rpm cultivates 36h.As shown in NO.5~6 SEQ ID, carrying out bacterium colony PCR can verify the primer sequence of PCR of the present invention
Single-swap recon, PCR product is through agarose gel electrophoresis, and the results are shown in Figure 2, sees whether have two length different
Purpose band.After the completion of bacterium colony PCR, single-swap recon is inoculated in the seed culture medium of non-resistant by the present invention, passage two
Secondary, passage of the present invention is preferably carried out in shaking table, and the temperature of the shaking table is preferably 25~35 DEG C, more preferably 28
~32 DEG C, most preferably 30 DEG C；The rotating speed of the shaking table is preferably 150~250rpm, more preferably 180~
220rpm, most preferably 200rpm.The time of passage of the present invention is preferably 18~30h, more preferably 22~
26h, most preferably for 24 hours.
After F2 is obtained for single-swap recon, the F2 is seeded to the TPG containing sucrose by the present invention for single-swap recon
On solid medium, 65~78h is cultivated, double exchange reorganization of phbB gene lists copy must be knocked out.Sucrose of the present invention exists
Mass concentration in TPG solid mediums is preferably 8~12%, more preferably 9~11%, most preferably 10%.Institute of the present invention
The time for stating culture is preferably 66~75h, more preferably 70~73h, most preferably 72h.
The present invention further includes preferably bacterium colony after double exchange reorganization for obtaining the knockout phbB gene lists copy
PCR verify, the bacterium colony PCR be preferably select knock out phbB gene lists copy the sub- single bacterium of double exchange reorganization drop down onto 5mL without
For 24 hours, the temperature of the culture is preferably 25~35 DEG C, more preferably 28~32 to shaking table culture in the seed culture medium of antibody
DEG C, most preferably 30 DEG C；The rotating speed of the shaking table is preferably 150~250rpm, more preferably 180~220rpm, most
Preferably 200rpm.The primer sequence of bacterium colony PCR of the present invention is as shown in NO.7~8 SEQ ID.Utilize SEQ ID
Primer shown in NO.5~6, double exchange reorganization can be verified by carrying out bacterium colony PCR, and PCR product is observed through agarose gel electrophoresis
Whether there is a shorter purpose band (500bp or so).
It obtains after knocking out double exchange reorganization that phbB gene lists copy, the present invention is copied with the knockout phbB gene lists
Double exchange reorganization be template, utilize the primer of NO.9~12 SEQ ID to repeat step a)~h), phbB Gene Doubles must be knocked out
Double exchange reorganization of copy.In the present invention, it when repeating the above steps, repeats step g) and step h) also includes preferably
There are bacterium colony PCR verifications, the bacterium colony PCR verifications are with primer sequence respectively such as SEQ ID NO:13~14 and SEQ ID NO:15~
PCR product is obtained shown in 16 every time there is a shorter purpose band through agarose gel electrophoresis observation being expected results.
Double exchange reorganization that techniques described above scheme of the present invention obtains is template, utilizes SEQ ID NO.17 successively
~20, the primer of NO.23~26 SEQ ID, NO.27~30 SEQ ID and NO.33~36 SEQ ID repeats step a)~h),
Step a)~h in 4 periods is repeated altogether) obtain the engineering bacteria T-3- Δs PHB for not producing poly- β hydroxybutyric acids.Primer of the present invention
Information is as shown in table 1：
1. primer information of table
After obtaining engineering bacteria T-3- Δs PHB, the present invention trains the engineering bacteria T-3- Δs PHB in TPG fluid nutrient mediums
It supports, carries out plasma mutagenesis, obtain mutagenesis engineering bacteria T-3- Δs PHB.TPG fluid nutrient mediums of the present invention include glucose, egg
White peptone, yeast powder and beef extract powder；The content of glucose is preferably 8~12g/L in the TPG fluid nutrient mediums, more preferably
For 9~11g/L, most preferably 10g/L.The content of peptone is preferably 3~7g/L in TPG fluid nutrient mediums of the present invention,
More preferably 4~6g/L, most preferably 5g/L.In TPG fluid nutrient mediums of the present invention the content of yeast powder be preferably 1~
5g/L, more preferably 2~4g/L, most preferably 3g/L.The content of beef extract powder is excellent in TPG fluid nutrient mediums of the present invention
It is selected as 1~5g/L, more preferably 2~4g/L, most preferably 3g/L.The present invention is to each component in the TPG fluid nutrient mediums
There is no particular determinations in source, utilize the conventional commercial product of this field.Culture of the present invention, preferred culture to work
The cell density of journey bacterium T-3- Δs PHB is 0.5 × 105~2 × 105A/mL, more preferably 0.8 × 105~1.2 × 105A/
ML, most preferably 1 × 105A/mL.
The process of plasma mutagenesis of the present invention includes preferably：Bacterium is added dropwise to sample injector bottom, utilizes MPMS instrument
Device carries out mutagenesis, obtains mutagenesis engineering bacteria.Dropwise addition of the present invention preferably aseptically carries out, more preferably in superclean bench
Middle progress.Sample injector of the present invention is preferably the sample injector of multi-functional plasma mutagenesis instrument, and the present invention is to the sample-adding
There is no particular determinations for method.The dripping quantity of bacterium of the present invention is preferably 15~25 μ L, more preferably 18~22 μ L, optimal
It is selected as 20 μ L.For the present invention after being added dropwise to complete, preferred includes being stored at room temperature, and time of the standing is preferably 8~
12min, more preferably 9~11min, most preferably 10min make bacterium solution form one layer of mycoderm in sample injector bottom.The present invention
The MPMS instruments are preferably the instrument after sterilizing, and there is no particular determinations for mode of the present invention to the sterilizing, preferably exist
Ultraviolet sterilization 30 minutes.When mutagenesis of the present invention, N2Throughput is preferably 8~15slm, more preferably 10~13slm, most
Preferably 12slm.The time of mutagenesis of the present invention is preferably 30~180s, more preferably 60~150s, most preferably 90s.
After obtaining mutagenesis engineering bacteria T-3- Δs PHB, the present invention trains the mutagenesis engineering bacteria T-3- Δs PHB in TPG solids
It supports and is cultivated on base, screen the highest bacterial strain of hydrogel yield, the highest bacterial strain of hydrogel yield is the engineering for producing hydrogel
Bacterium NXdP.Further include preferably by mutagenesis engineering bacteria T-3- Δs PHB weights before the present invention cultivates on TPG solid mediums
It is outstanding.It is of the present invention to be resuspended preferably by the obtained mutagenesis engineering bacteria and fresh TPG fluid nutrient mediums according to 1:50
Volume ratio mixing, obtain the TPG suspension of mutagenesis engineering bacteria.Culture of the present invention preferably applies 100 μ L suspension
On cloth to TPG solid mediums, the temperature of the culture is preferably temperature and is preferably 25~35 DEG C, more preferably 28~
The time of 32 DEG C, most preferably 30 DEG C, the culture is preferably 1~5d, more preferably 2~4d, most preferably 3d.
The content of agar powder described in TPG solid mediums of the present invention is preferably 15g/L.The process of screening of the present invention is excellent
Choosing packet is screened by forming the size of bacterium colony after culture same time, and in the present invention, bacterium colony is bigger, and hydrogel yield is higher.
The engineering bacteria NXdP production water-settings built the present invention also provides above-mentioned engineering bacteria NXdP or using the above method
The method of glue, includes the following steps：
1. engineering bacteria NXdP single bacterium colonies are seeded in TPG fluid nutrient mediums, 20~26h of shake culture obtains culture solution；
2. the 1. culture solution that step obtains is seeded in seed culture medium, 20~26h of shake culture obtains seed
Liquid；
3. by step, 2. the seed liquor is inoculated in fermentation medium, and ferment 68~75h, obtains zymotic fluid；
4. the 3. zymotic fluid that step obtains is diluted with distilled water, heating centrifugation, detaches supernatant liquid and thalline is heavy
It forms sediment；
5. the 4. supernatant liquid pH to 3.0 that regulating step obtains, collects the precipitation of generation, obtains hydrogel, be named as
Declare good fortune glue.
When utilizing works bacterium NXdP production hydrogels, engineering bacteria NXdP single bacterium colonies are seeded to TPG fluid nutrient mediums by the present invention
In, 20~26h of shake culture obtains culture solution.NXdP single bacterium colonies of the present invention and the inoculation relationship of TPG fluid nutrient mediums are preferred
For 1/3~8mL, more preferably 1/4~6mL, most preferably 1/5mL.The temperature of shake culture of the present invention
Preferably 28~35 DEG C, more preferably 29~32 DEG C, most preferably 30 DEG C.The time of shake culture of the present invention is preferred
It is 22~25h, more preferably 23~24.5h, most preferably for 24 hours.
After obtaining culture solution, the culture solution is seeded in seed culture medium by the present invention, and 20~26h of shake culture must be planted
Sub- liquid.The volume ratio of culture solution and seed culture medium of the present invention is preferably 1：(20~60) are carried out when using fask oscillating method
When fermentation, the volume ratio is 1:20；When being fermented using batchwise, the volume ratio is 1:60.Institute of the present invention
The temperature for stating shake culture is preferably 28~35 DEG C, more preferably 29~32 DEG C, most preferably 30 DEG C.Shake of the present invention
The time for swinging culture is preferably 22~25h, more preferably 23~24.5h, most preferably for 24 hours.
After obtaining seed liquor, the seed liquor is inoculated in fermentation medium by the present invention, and ferment 68~75h, obtains zymotic fluid.
The inoculation volume of seed liquor of the present invention is preferably the 3~10% of the fermentation medium volume, and more preferably 4~6%, most
Preferably 5%.The ingredient of fermentation medium includes preferably glucose, beancake powder, K2HPO4, MgSO4And NaNO3, in the hair
In ferment culture medium, when being fermented using fask oscillating method, the concentration of the glucose is preferably 30~70g/L, more preferably 40
~50g/L, most preferably 40g/L；The concentration of the beancake powder is preferably 0.5~2g/L, more preferably 1~1.8g/
L, most preferably 1.54g/L；The K2HPO4Concentration be preferably 0.5~2g/L, more preferably 0.8~1.2g/L, most
Preferably 0.9g/L；The MgSO4Concentration be preferably 0.1~1g/L, more preferably 0.3~0.5g/L, most preferably
It is 0.4g/L；The NaNO3Concentration be preferably 1~2g/L, more preferably 1.2~1.8g/L, most preferably
1.54g/L；When being fermented using batchwise, the concentration of the glucose is preferably 30~50g/L, more preferably 40
~50g/L, most preferably 40g/L；The concentration of the beancake powder is preferably 0.5~2g/L, more preferably 1~1.8g/
L, most preferably 1.54g/L；The K2HPO4Concentration be preferably 0.5~2g/L, more preferably 0.8~1.2g/L, most
Preferably 0.9g/L；The MgSO4Concentration be preferably 0.1~1g/L, more preferably 0.3~0.5g/L, most preferably
It is 0.4g/L；The NaNO3Concentration be preferably 1~2g/L, more preferably 1.2~1.8g/L, most preferably
1.54g/L.The present invention is not particularly limited the source of each component in the fermentation medium, utilizes the conventional commercial of this field
Product.The temperature of fermentation of the present invention is preferably 28~35 DEG C, more preferably 29~32 DEG C, most preferably 30
℃.The time of fermentation of the present invention is preferably 69~74h, more preferably 70~73h, most preferably 72h.
After obtaining zymotic fluid, the present invention dilutes the zymotic fluid with distilled water, and heating centrifugation detaches supernatant liquid and bacterium
Body precipitates.Dilution of the present invention preferably 5~10 times of dilution, more preferably 6~8 times, most preferably 7 times.This hair
The temperature of the bright heating is preferably 95~121 DEG C, more preferably 99~110 DEG C, most preferably 105 DEG C；It is described to add
The time of heat is preferably 15~25min, more preferably 18~22min, most preferably 20min.Centrifugation of the present invention
Rotating speed be preferably 10000~15000rpm, more preferably 11000~13000rpm, most preferably 12000rpm, the centrifugation
Time be preferably 10~50min, more preferably 20~40min, most preferably 30min.
After obtaining supernatant liquid, the present invention adjusts the supernatant liquid pH to 3.0, collects the precipitation of generation, obtains hydrogel, orders
Entitled a surname's good fortune glue.Supernatant liquid pH of the present invention preferably utilizes dilute hydrochloric acid to adjust, and the concentration of the dilute hydrochloric acid is preferably
1mol/L.Precipitation of the present invention preferably after NaOH solution adjusts pH to neutrality, dries to obtain hydrogel.It is of the present invention
The concentration of NaOH solution is preferably 1mol/L.The condition of drying of the present invention is preferably 60 DEG C overnight.
Engineering bacteria NXdP provided by the invention and its construction method and application are carried out specifically with reference to embodiment
It is bright, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
The structure of the key gene knockout carrier of poly- β hydroxybutyric acids route of synthesis：
According to flow chart shown in FIG. 1, the genome of T-3 is extracted using extracts kit, the upstream and downstream of target gene is homologous
Arm uses primer SEQ ID NO respectively using T-3 genomes as template：1~4 and PrimeSTARDNA polymerases are expanded.It is logical
It crosses overlap PCR to connect upstream and downstream DNA fragmentation, by product by electrophoresis detection, and target gene band be returned by glue
It receives kit and carries out purifying recycling, obtain recombinant fragment.By recombinant fragment and pLO3 plasmids simultaneously using restriction enzyme SacI and
XbaI carries out digestion, 37 DEG C, 90min, the segment after digestion is carried out PCR purifying recycling using kit, by the recycling of the two
Product, in 16 DEG C of connections overnight, obtains recombinant plasmid pLO3- Δ gene, and recombinant plasmid is transferred to T4DNA ligases
The amplification of recombinant plasmid is carried out in E.coli S17 competent cells, picking is sequenced correct single bacterium colony and carries out glycerine preservation.
Embodiment 2
The structure of the key gene knockout carrier of poly- β hydroxybutyric acids route of synthesis：
According to flow chart shown in FIG. 1, the genome of T-3 is extracted using extracts kit, the upstream and downstream of target gene is homologous
Arm uses primer SEQ ID NO respectively using T-3 genomes as template：1~4 and PrimeSTARDNA polymerases are expanded.It is logical
It crosses overlap PCR to connect upstream and downstream DNA fragmentation, by product by electrophoresis detection, and target gene band be returned by glue
It receives kit and carries out purifying recycling, obtain recombinant fragment.By recombinant fragment and pLO3 plasmids simultaneously using restriction enzyme SacI and
PacI carries out digestion, 37 DEG C, 90min, the segment after digestion is carried out PCR purifying recycling using kit, by the recycling of the two
Product, in 16 DEG C of connections overnight, obtains recombinant plasmid pLO3- Δ gene, and recombinant plasmid is transferred to T4DNA ligases
The amplification of recombinant plasmid is carried out in E.coli S17 competent cells, picking is sequenced correct single bacterium colony and carries out glycerine preservation.
Embodiment 3
The structure of poly- β hydroxybutyric acids engineering strain is not produced：
T-3 single bacterium colonies are chosen in TPG solid mediums to be seeded to containing Cmr5ml seed culture mediums, in 30 DEG C of constant-temperature tables
200rpm is cultivated for 24 hours, and the single bacterium of picking E.coli s17/pLO3- Δs gene is dropped down onto containing tetrLB liquid medium in, in
200rpm cultivates 8h in 37 DEG C of constant-temperature tables, and two plants of bacterium respectively take 5ml 6000rpm centrifugations 5min to collect thalline, with 10mmol/L's
MgSO4Solution washes twice, centrifugation, then with the MgSO of 200ul4Thalline is resuspended in solution, by T-3 and E.coli s17/pLO3- Δs
Gene is according to 2:It filters after 1 ratio mixing and is placed on the filter membrane of 0.22um to aperture, the filter membrane of thalline upward is placed in nothing
In resistant panel, culture 12h carries out engagement transfer in 30 DEG C of constant incubators.With 200ul MgSO4Solution washs on filter membrane
Thalline, be coated on containing Cm after gradient dilutionrAnd tetrDual anti-tablet on, cultivate 72h in 30 DEG C of constant incubators.?
Picking single bacterium drops down onto 5ml containing in dual anti-seed fluid nutrient mediums of saccharomycete on dual anti-tablet, and 200rpm is cultivated in 30 DEG C of constant-temperature tables
36h.Bacterium colony PCR, which is carried out, using primer SEQ ID NO.5~6 verifies single-swap recon, PCR product agarose gel electrophoresis,
It sees whether with purpose band.Single-swap recon is inoculated in the 5ml seed culture mediums of non-resistant, 30 DEG C of constant-temperature tables
For 24 hours, passage twice, is then applied on 10% sucrose plate and is cultivated in 30 DEG C of constant incubators for middle 200rpm cultures
72h.Picking single bacterium drops down onto in 5ml test tubes, 30 DEG C, and 200rpm is cultivated for 24 hours, and bacterium colony is carried out using primer SEQ ID NO.7~8
PCR is verified, determine correct double exchange reorganization, is preserved and is carried out next step and continue to knock out, until obtaining not producing poly- β completely
The engineering strain of hydroxybutyric acid, knocking out phb gene copies every time, the results are shown in Figure 2, is finally named as T-3- Δs
PHB, glycerol tube are stored in -80 DEG C.
Embodiment 4
Plasma mutagenesis is carried out using multi-functional plasma mutagenesis system (MPMS)：
Before plasma mutagenesis, by T-3- Δ PHB bacterial strains in TPG culture mediums preculture to about 105The cell of/mL is close
Degree；20 μ L bacterial suspensions are dropped in the sample injector bottom of multi-functional plasma mutagenesis instrument in superclean bench；Then in room temperature
It is lower to stand 10 minutes, form one layer of mycoderm in sample injector bottom.Use previously 30 minutes MPMS instruments pair of ultraviolet sterilization
Bacterium carries out mutagenesis.When carrying out mutagenesis, N2Throughput is fixed as 12slm, according to different time spans (30s-180s), uses gas
Stream impact cell simultaneously carries out gradient mutagenesis.Cell is resuspended in the fresh TPG fluid nutrient mediums of 1mL, and each is mixed and is hanged
Supernatant liquid (100 μ L) is applied on two TPG tablets, is then cultivated 3 days at 30 DEG C.Larger bacterium colony is selected to carry out shake flask fermentation
Compare the yield of its hydrogel.Finally obtain engineered strain NXdP.
Embodiment 5
Genetic engineering bacterium NXdP fask oscillating method fermenting and producings hydrogel (a surname's good fortune glue)：
1. genetic engineering bacterium NXdP single bacterium colonies are seeded in the TPG fluid nutrient mediums of 5ml, 30 DEG C of shaken cultivations 24 are small
When；
2. culture solution made from step (1) is seeded in the seed culture medium of 100ml, 30 DEG C of shaken cultivations 24 hours；
3. seed liquor made from step (2) is seeded in glass shaking flask with 6~10% inoculum concentration, 30 DEG C, constant pH
6.8~7.2, it cultivates 72 hours；
4. by the zymotic fluid of step (3) distilled water at least 5 times of volume dilutions, 105 DEG C are heated 20 minutes, high speed centrifugation point
From zymotic fluid and bacterial sediment；
5. the dilute hydrochloric acid of the zymotic fluid in step (4) is adjusted pH to 3.0 or so, precipitation is adjusted to pH neutrality through NaOH,
Drying obtains biological polyoses, and sterling yield is 15.03 ± 2.35g/L, and total output is 32.51 ± 3.18g/L, and conversion ratio is
48.8%.
Embodiment 6
Genetic engineering bacterium NXdP batchwises produce hydrogel (a surname's good fortune glue)：
1. genetic engineering bacterium NXdP single bacterium colonies are seeded in the TPG fluid nutrient mediums of 5ml, 30 DEG C of shaken cultivations 24 are small
When；
2. culture solution made from step (1) is seeded in the seed culture medium of 300ml, 30 DEG C of shaken cultivations 24 hours；
3. seed liquor made from step (2) is seeded in 5L fermentation tanks with 6~10% inoculum concentration, 30 DEG C, constant pH
6.8~7.2, it cultivates 72 hours；
4. by the zymotic fluid of step (3) distilled water at least 5 times of volume dilutions, 105 DEG C are heated 20 minutes, high speed centrifugation point
From zymotic fluid and bacterial sediment；
5. the dilute hydrochloric acid of the zymotic fluid in step (4) is adjusted pH to 3.0 or so, precipitation is adjusted to pH neutrality through NaOH,
Drying obtains biological polyoses, and sterling yield is 21.20 ± 0.38g/L, and total output is 32.03 ± 2.21g/L, and conversion ratio is
80%.
Experimental example 1
The measurement of batch fermentation different times glucose content：
Using bio-sensing analyzer SBA-40C (the Shandong Province's biosensor emphasis realities for being equipped with glucose enzyme membrane circle
Test room) measure zymotic fluid in glucose content.After fermentation, zymotic fluid is carried out to the dilution of 5 times of volumes with distilled water,
100 DEG C are heated 10 minutes, and supernatant precipitation is centrifuged.Supernatant is used to measure the content of glucose in zymotic fluid.With
The glucose of 100mg/dl is as titer, and measurement result is as shown in figure 3, result is shown：It is residual in different fermentations period zymotic fluid
The glucose content stayed is differed from 40g/L to 0g/L, and as fermentation time successively decreases, it was demonstrated that the high conversion rate of glucose.
Experimental example 2
The measurement of batch fermentation different times fermentation broth viscosity：
Viscosimetric analysis, using 64# rotors, 60 revs/min are carried out using U.S. Brookfield viscometer DV_II+
Viscosity is measured under the conditions of clock.Measurement result is as shown in Figure 3b, and after measured, when culture was to 40 hours, fermentation broth viscosity starts to increase
Add, it is about 6.4pa.s that culture reaches maximum value to fermentation broth viscosity at 72 hours, it was demonstrated that the purity of a surname's good fortune glue is high.
Experimental example 3
Declare the measurement of good fortune glue rheologic behavio(u)r：
The rheological behavior of a surname's good fortune glue is measured by TA rheometers, by a surname's good fortune peptization (10mg/ml) in ultra-pure water, inspection
Survey condition is 25 DEG C, and testing result is as shown in figure 4, range of shear rate is 0.001s-1-1000s-1。
Experimental example 4
Engineering strain fermenting and producing declares good fortune glue to be compared with wild-type strain：Using ferment tank, except bacterial strain is different
Outside, remaining condition all same, the results are shown in Table 2 for detection：
In conclusion the genetic engineering bacterium NXdP constructed by the present invention can effectively improve transparent aquagel (a surname's good fortune glue)
Yield, and there is very high carbohydrate gum conversion ratio, which has extensive prospects for commercial application.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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Claims (10)
1. the deposit number of the engineering bacteria NXdP, the engineering bacteria NXdP of one plant of production hydrogel are CGMCC15406, preservation place
For China General Microbiological culture presevation administrative center, the preservation time is on 03 01st, 2018.
2. the method for engineering bacteria NXdP, includes the following steps described in a kind of structure claim 1：
1) double crossing over methods of homologous recombination is utilized to knock out the phbB genes of Sphingol single-cell (Sphingomonas sp.T-3),
Obtain the engineering bacteria T-3- Δs PHB for not producing poly- β hydroxybutyric acids；
2) the engineering bacteria T-3- Δs PHB that step 1) obtains is cultivated in TPG fluid nutrient mediums, plasma is carried out after culture
Mutagenesis obtains mutagenesis engineering bacteria T-3- Δs PHB；The TPG fluid nutrient mediums include：8~12g/L of glucose, 3~7g/ of peptone
1~5g/L of L, 1~5g/L of yeast powder and beef extract powder；
3) the mutagenesis engineering bacteria T-3- Δs PHB that step 2) obtains is cultivated on TPG solid mediums, screening hydrogel production
Highest bacterial strain is measured, the hydrogel yield highest bacterial strain is the engineering bacteria NXdP for producing hydrogel；The TPG solid mediums packet
It includes：8~12g/L of glucose, 3~7g/L of peptone, 1~5g/L of yeast powder, 1~5g/L of beef extract powder and agar powder 15g/L.
3. according to the method described in claim 2, it is characterized in that, step 1) the double crossing over methods of homologous recombination, including with
Lower step：
A) using Sphingol single-cell T-3 genomic DNAs template, SEQ ID NO are utilized：The primer of sequence shown in 1~4 carries out weight
Recombinant fragment is recycled to obtain in folded PCR, purifying；
B) recombinant fragment and pLO3 plasmids described in restriction enzyme SacI and XbaI or SacI and PacI difference double digestions are utilized,
Digestion products are attached, recombinant plasmid pLO3- Δs gene is obtained；
C) the recombinant plasmid pLO3- Δs gene that step b) is obtained is transformed into E.Coli S17 competent cells, is obtained
E.Coli S17/pLO3-Δgene；
D) single bacterium colony of the obtained E.Coli S17/pLO3- Δs gene of step c) is being contained into tetrLB liquid medium
E.Coli S17/pLO3- Δ gene thalline are collected by centrifugation in 6~10h of middle culture；
E) Sphingol single-cell T-3 single bacterium colonies are selected to be inoculated into containing CmrSeed culture medium in, cultivate 20~28h, be collected by centrifugation
Sphingol single-cell T-3 thalline；
F) by the E.Coli S17/pLO3- Δ gene thalline that step d) is obtained and the Sphingol single-cell T-3 bacterium that step e) is obtained
Body uses MgSO respectively4After solution is resuspended, according to 1：The volume ratio mixing of (1.5~2.5) filters, and is connect on TPG solid mediums
10~15h of transfer is closed, single-swap recon is obtained；
G) the single-swap recon that step f) is obtained is inoculated in the seed culture medium of non-resistant, passage twice, obtains F2
For single-swap recon；The seed culture medium includes：8~12g/L of sucrose, 1.8~3g/L of peptone, 1~2.5g/ of yeast powder
L, K2HPO41.8~3g/L and MgSO40.06~0.15g/L；
H) F2 that step g) is obtained is seeded to for single-swap recon on the TPG solid mediums containing sucrose, culture 65
~78h must knock out double exchange reorganization of phbB gene lists copy；
I) double exchange reorganization of the knockout phbB gene lists copy obtained using step h) utilizes SEQ ID as template
The primer of sequence shown in NO.9~12 repeats step a)~h), double exchange reorganization of phbB Gene Doubles copy must be knocked out；
J) using it is described knockout phbB Gene Doubles copy double exchange reorganization as template, successively utilize NO.17~20 SEQ ID,
The primer of NO.23~26 SEQ ID, NO.27~30 SEQ ID and NO.33~36 SEQ ID repeats step a)~h), it obtains not
Produce the engineering bacteria T-3- Δs PHB of poly- β hydroxybutyric acids.
4. according to the method described in claim 3, it is characterized in that, the program of the step a) over-lap PCRs, including：1st~10
A cycle, 98 DEG C of denaturation 10s are warming up to 72 DEG C of annealing, 72 DEG C of extension 1min for 55 DEG C in 15s；11st~20 cycle, 98 DEG C
It is denaturalized 10s, 55 DEG C of annealing, 72 DEG C of extension 2min are cooled to for 72 DEG C in 15s；21st~31 cycle, 98 DEG C denaturation 10s, 60
DEG C annealing 15s, 72 DEG C extension 2min；32nd cycle, 72 DEG C of extension 10min.
5. according to the method described in claim 3, it is characterized in that, matter of the step h) sucrose in TPG solid mediums
Measure a concentration of 8~12%.
6. the engineering bacteria that engineering bacteria NXdP described in claim 1 or claim 2~6 any one of them method are built
Applications of the NXdP in producing hydrogel.
7. application according to claim 6, which is characterized in that include the following steps：
1. the engineering bacteria NXdP single bacterium colonies are seeded in TPG fluid nutrient mediums, 20~26h of shake culture obtains culture solution；
2. the 1. culture solution that step obtains is seeded in seed culture medium, 20~26h of shake culture obtains seed liquor；
3. by step, 2. the seed liquor is inoculated in fermentation medium, and ferment 68~75h, obtains zymotic fluid；
4. the 3. zymotic fluid that step obtains is diluted with distilled water, heating centrifugation, isolated supernatant liquid and thalline are heavy
It forms sediment；
5. the 4. supernatant liquid pH to 3.0 that regulating step obtains, collects the precipitation of generation, obtains hydrogel, be named as Xuan Fu
Glue.
8. application according to claim 7, which is characterized in that the step 1. temperature with the step 2. shake culture, solely
Vertical is 28~35 DEG C.
9. application according to claim 7, which is characterized in that the step 3. fermentation medium, including：Glucose 30~
70g/L, beancake powder 0.5~2g/L, K2HPO41~2g/L, MgSO40.1~1g/L and NaNO31~2g/L.
10. application according to claim 7, which is characterized in that step 3. the fermentation temperature be 28~35 DEG C.
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