CN103290048B - Plasmid and engineering bacteria suitable for bacillus homologous recombination - Google Patents

Abstract

The invention relates to a plasmid and engineering bacteria suitable for bacillus homologous recombination. The plasmid and engineering bacteria comprise linear segments of a sequence 8; the plasmid takes a sequence of 16SrDNA as homologous arms; a Kanamycin resistance selection marker gene, a P43 promoter, a multiple-cloning site, and a terminator sequence are formed between the homologous arms. By adopting the plasmid disclosed by the invention, various target genes can be integrated on each bacillus inoculum; a converter containing different copy numbers of target genes can be obtained; stable and efficient expression of the target genes in the bacillus is achieved. In addition, pKD314-apr4M recombinant plasmids are built by utilizing the plasmid; different copy numbers of stable and high-yield engineering bacteria are obtained by a homologous recombination method.

Description

A kind of plasmid and engineering bacteria being applicable to bacillus homologous recombination

Technical field

The invention belongs to genetically engineered field, relate to a kind of plasmid and the engineering bacteria that are applicable to bacillus homologous recombination.

Technical background

Genus bacillus is secretory dna engineering Host Strains.Research shows, a lot of foreign protein also achieves secreting, expressing (table 1) in subtilis, therefore, produces foreign protein more and more by people are paid close attention to subtilis.

The recombinant protein that table 1 is expressed in subtilis

Genus bacillus has following advantage as the expression system of foreign gene: subtilis has a set of efficient secreting signal peptide and chaperone systems, thus target protein can be secreted efficiently, also simplify the purification procedures of target protein, and as a rule, the recombinant heterologous recombinant protein of secreted from bacillus has native conformation and biological activity; Genus bacillus strictly aerobic, growth rapidly, simple, the large scale fermentation technology maturation of culture condition; Most genus bacillus use safety, no pathogenicity; Codon is without preferences, and transcription and translation mechanism, genetic background are clearer.

At present the genetic modification of the bacillus such as subtilis is mainly concentrated on and realize free expression by heredity importing expression plasmid, but unstable by the engineering bacteria hereditary property of this kind of method structure.Need to add microbiotic in process of production and prevent plasmid loss, be difficult to spread in industrial production.In addition, adopt methods of homologous recombination to be incorporated on karyomit(e) by expressing gene, but a copy number can only be integrated, and homologous recombination efficiency is lower, DeGrain.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art part, a kind of plasmid and the engineering bacteria that are applicable to bacillus homologous recombination are provided, various goal gene can be incorporated on the various bacterium of bacillus by plasmid of the present invention, and the transformant that can obtain containing different copy number goal gene, realize goal gene and express at the stability and high efficiency of bacillus

The object of the invention is to be achieved through the following technical solutions:

Be applicable to a plasmid for bacillus homologous recombination, comprise the linear fragment of sequence 8.

And described plasmid, using the sequence of 16SrDNA as homology arm, is card that resistance screening marker gene, P43 promotor, multiple clone site, terminator sequence between homology arm.

And the linear fragment of described sequence 8 is connected to the pstI restriction enzyme site of plasmid pMD18Tsimple-16SrDNA.

A kind of bacillus engineering bacteria, utilizes the plasmid construction being applicable to bacillus homologous recombination to obtain.

One strain Alkaliphilic bacillus engineering bacteria, utilizes the plasmid being applicable to bacillus homologous recombination, multiple copied goal gene apr4M is incorporated into the engineering bacteria on karyomit(e) 16SrDNA.

Advantage of the present invention and positively effect as follows:

1, the present invention is at analysis 16SrDNA sequence high conservative, copy number high and have feedback regulation effect (insert lethal one or several copy 16SrDNA, the total amount of 16SrRNA remains unchanged, the normal growth metabolism of thalline can not be affected movable) characteristic basis on, design and construct and be provided by the inventionly applicable to bacillus homologous recombination plasmid, solve by free improvement bacterial classification hereditary property of expressing unstable and be difficult to the problem that to be incorporated into the form of multiple copied by expressing gene by methods of homologous recombination on karyomit(e), this plasmid is utilized successfully to construct Alkaliphilic bacillus engineering bacteria containing different copy number, the unit cell yield of enzyme of constructed engineering bacteria comparatively wild-type improves about 278%.

2, various goal gene can be incorporated on the various bacterium of bacillus by plasmid of the present invention, and can obtain the transformant containing different copy number goal gene, realizes goal gene and expresses at the stability and high efficiency of bacillus.In addition, utilize this plasmid construction pKD314-apr4M recombinant plasmid, and obtained the engineering bacteria stablizing high yield containing different copy number by methods of homologous recombination.

Accompanying drawing explanation

Fig. 1 is homologous recombination vector pKD314 structure iron in the present invention;

Fig. 2 is different copy number goal gene primary dcreening operation result constructed by the present invention;

Fig. 3 is recombinant plasmid pKD314-apr4PCR proof diagram constructed by the present invention;

The nucleic acid electrophoresis figure wherein M:DNAMarker1,2 of PCR checking in Fig. 4 bacterial strain constructed by the present invention: homologous recombination success, 3: not reorganized;

Fig. 5 surveys by the present invention the typical curve of enzyme activity determination.

Embodiment

Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.

Being applicable to the plasmid of bacillus homologous recombination: using the sequence of 16SrDNA as a homology arm, is card that resistance screening marker gene, P43 promotor, multiple clone site, terminator sequence between homology arm.

One strain Alkaliphilic bacillus engineering bacteria, utilizes the plasmid pKD314-apr4M built by pKD314 multiple copied goal gene to be incorporated into engineering bacteria on karyomit(e) 16SrDNA.

Structure and the detecting step of above-mentioned homologous recombination plasmid and Alkaliphilic bacillus engineering bacteria are:

One, pMD18Tsimple-16SrDNA plasmid is built.

Two, kan resistant gene is connected in complete sequence synthesis expression regulation original paper psg(P43 promotor, multiple clone site, terminator sequence) on, build kan-psg.The concrete sequence of expression regulation original paper psg is shown in sequence 7.

Three, be connected in kan-psg linear fragment by pMD18Tsimple-16SrDNA plasmid, be built into homologous recombination plasmid pKD314, pKD314 concrete structure is shown in Fig. 1.

Four, Alkaliphilic bacillus Sumizyme MP mature peptide encoding gene apr4 is connected in pKD314 multiple clone site is built into pKD314-apr4 plasmid.

Five, obtain linear fragment with ECORV digestion with restriction enzyme recombinant plasmid pKD314-apr4M, linear fragment electricity is proceeded to Alkaliphilic bacillus.

Six, engineering bacteria and original bacteria unit cell enzymatic productivity is compared.

The detailed step of the structure of bacterial strain of the present invention is as follows:

One, the structure of homologous recombination vector

Two, the amplification of 1. each gene

(1) the gene amplification of genus bacillus 16SrDNA

With Alkaliphilic bacillus (through the homology of the whole bacillus 16SrDNA of sequence alignment more than 95%, therefore the genome of choosing a kind of bacterium is wherein as template) carry out PCR for template, then by agarose gel electrophoresis and cut glue and reclaim and obtain goal gene fragment.Required primer is as follows:

P15′-CGGATATCGAGAGTTTGATCCTGGCTGGCTCAG-3′(ECORV)

P25′-CGGATATCAAGGAGGTGATCCAGCCGCA-3′(ECORV)

Reaction system is 50 μ L, and proportioning is as follows:

PCR program is as follows:

95 DEG C of denaturation 5min; 94 DEG C of sex change 45s; 57 DEG C of annealing 45s; 72 DEG C extend 1min30s; 30 rear 72 DEG C of extension 10min of circulation; 4 DEG C of preservations.

(2) the amplification of resistant gene (kan) received by card

With plasmid pBE2 for template carries out PCR, then by agarose gel electrophoresis and cut glue reclaim obtain goal gene fragment.Required primer is as follows:

P35′-CGGACTAGTGCCGATGAAGATGGATTTTCTATTA-3′(SpeI)

P45′-GGGACTAGTGCGCCCCGGGTTCGAAGGGCA-3′(SpeI)

Reaction system is 50 μ L, and proportioning is as follows:

PCR program is as follows:

95 DEG C of denaturation 5min; 94 DEG C of sex change 45s; 58 DEG C of annealing 45s; 72 DEG C extend 1min30s; 30 rear 72 DEG C of extension 10min of circulation; 4 DEG C of preservations.

(3) the amplification of mature peptide gene (apr4)

First, with Alkaliphilic bacillus genome for template carries out PCR, then by agarose gel electrophoresis and cut glue reclaim obtain goal gene fragment.Required primer is as follows:

P55′-CGCGGATCCCAATCAGTGCCATGGGGAAT-3′(BamHI)

P65′-CCCAAGCTTTTAGCGTGTTGCCGCTTC-3′(HindIII)

Reaction system is 50 μ L, and proportioning is as follows:

PCR program is as follows:

95 DEG C of denaturation 5min; 94 DEG C of sex change 45s; 58 DEG C of annealing 45s; 72 DEG C extend 1min30s; 30 rear 72 DEG C of extension 10min of circulation; 4 DEG C of preservations.

2. the structure of general recombinant vectors pKD314, building process is as follows

(1) after being connected with 16SrDNA gene by plasmid pMD18Tsimple, obtain plasmid pMD18Tsimple-16SrDNA.

(2) search P43 promoter sequence according to NCBI (GenBankaccessionNo.K02174.1) and (GenBankaccessionNo.AAA22212) searches terminator sequence, according to the sequence of the sequences Design synthesis of report, between promotor and terminator, introduce multiple clone site.Concrete sequence psg is shown in sequence 7.Sequent synthesis entrusts invitrogen company.The plasmid called after pUC57-psg of synthesis.

(3) the SpeI restriction enzyme site linking pUC57-psg after being cut by that resistant gene of card kan restriction enzyme SpeI enzyme obtains plasmid pUC57-kan-psg.

(4) plasmid pUC57-kan-psg restriction enzyme pstI enzyme is cut and is obtained linear fragment (linear fragment is shown in sequence 8), this linear fragment is connected on plasmid pMD18Tsimple-16SrDNA and obtains plasmid pKD314, wherein plasmid pMD18Tsimple-16SrDNA also needs to cut through pstI enzyme, and then connects.

Above vector construction process ligase enzyme used is T4DNA ligase enzyme, and linked system is 20 μ L, and linked system is as follows: goal gene: carrier (ratio of volumetric molar concentration)=3:1-9:1, adds the mixing of appropriate 0.2ul ligase enzyme, under 16 ° of C, and reaction overnight.

The enzyme system of cutting is 50 μ L, and concrete proportioning is.

37 DEG C of enzymes cut 3h, add 10 × LoadingBuffer5 μ L termination reaction that restriction enzyme is subsidiary in digestion products.1% agarose gel electrophoresis detects digestion products.

3. the structure of high yield alkali protein Alkaliphilic bacillus

(1) the structure of recombinant plasmid pKD314-apr4M

PKD314 is connected with Sumizyme MP mature peptide gene apr4 after BamHI with HindIII double digestion, obtains recombinant plasmid pKD314-apr4.

Vector construction process ligase enzyme used is T4DNA ligase enzyme, and linked system is 20 μ L, and linked system is as follows: goal gene: carrier (ratio of volumetric molar concentration)=3:1-9:1, adds the mixing of appropriate 0.2ul ligase enzyme, under 16 ° of C, and reaction overnight.

ECORV double digestion recombinant plasmid obtains homologous recombination linear fragment.The enzyme system of cutting is 50 μ L.

37 DEG C of enzymes cut 3h, add 10 × LoadingBuffer5 μ L termination reaction that restriction enzyme is subsidiary in digestion products.1% agarose gel electrophoresis detects digestion products, to the linear fragment cutting glue recovery 4.3kb.Plasmid PCR proof diagram is shown in Fig. 2.

(2) the electricity of recombinant fragment turns

After frozen Alkaliphilic bacillus competent cell (TCCC11263) is dissolved on ice, get 50 μ L add restructuring linear fragment mix, then transfer in the electric shock cup of ice precooling.After using this cell mixture electricimpulse of high-voltage pulse electric shock conversion electric shock to terminate, rapidly cell suspension is transferred in the EP pipe of room temperature recovery medium, shaking culture for some time, get 150 μ L conversion fluids after the serial dilution of recovery wild Oryza species and be coated with 1% skim milk plate (kantlex containing 30 μ g/mL) screening positive clone.The results are shown in Figure 3.

(3) the checking of recombinant conversion

Select the single bacterium colony in resistant panel, be inoculated in containing in 40 μ g/mL kantlex LB substratum, 32 DEG C of shaking tables cultivate 24 hours, extract genome.

PCR verifies, checking primer is P1, P2.

Reaction system is 20 μ L, and proportioning is as follows:

PCR program is as follows:

95 DEG C of denaturation 5min; 94 DEG C of sex change 45s; 61 DEG C of annealing 45s; 58 DEG C extend 1min30s; 30 rear 72 DEG C of extension 10min of circulation; 4 DEG C of preservations.

The result is shown in Fig. 4.

(4), unicellular recombinant bacterium enzymatic productivity measures

Get in the bacterium single bacterium colony access 25mL seed culture medium on fresh plate, 34 DEG C, 180r/min constant-temperature table cultivation 12h; By in the inoculum size access 50mL fermention medium of 1%, identical culture condition cultivates 48h.Fermented liquid in 4 DEG C, 8000r/min is centrifugal, supernatant liquor is crude enzyme liquid.Centrifugal obtained thalline sterilized water suspends, and surveys OD ₆₀₀.

With light industry ministerial standard QB/T1803-93(Folin reagent color developing method): 1mL enzyme liquid is at 40 DEG C, and the enzyme amount required for pH10.5, per minute reaction generation 1 μ g tyrosine is 1 enzyme activity unit (U/mL).

1. the drafting of typical curve

The tyrosine standardized solution (0,10,20,30,40,50,60 μ g/mL) of preparation different concns.Get tyrosine standardized solution 1.00mL, add the sodium carbonate solution 5.00mL of 0.4mol/L, Folin reagent solution 1.00mL, be placed in 40 ± 0.2 DEG C of water-baths and develop the color 20min.Take out, measure its absorbance A 680(respectively not contain the pipe of tyrosine for blank with spectrophotometer).With absorbance A 680 for ordinate zou, the concentration c of tyrosine is X-coordinate, drawing standard curve (this line should pass through zero point).

2. crude enzyme liquid borax-NaOH(pH10.5) buffered soln is diluted to proper concn, as enzyme liquid to be measured.

Borax/NaOH(pH10.5) casein solution of buffer 1% is as substrate and crude enzyme liquid is diluted suitable multiple.Get the enzyme liquid of 1mL dilution, 40 DEG C of insulation 2min, add the substrate 1mL of same temperature, in 40 DEG C of reaction 10min, add 2mL10% trichoroacetic acid(TCA) termination reaction.Leave standstill centrifugal, get 1mL supernatant liquor, add 5mL0.4mol/LNa ₂cO ₃solution, 1mL forint phenol reagent, mixing, 40 DEG C of insulation 20min, measure absorbance A 680.With inactivator liquid for contrast.

Enzyme method of calculation alive

X=A×K×4/10×n＝0.4×A×K×n

In formula: the enzyme activity (U/g or U/mL) of X-sample

The mean light absorbency of A-sample parallel test

K-extinction constant

The cumulative volume (mL) of 4-reaction reagent

10-reaction times

N-extension rate

The light absorption value of the tyrosine standardized solution of different concns and the numerical relation of tyrosine concentration are as shown in Figure 5.

Take tyrosine concentration as X-coordinate, the light absorption value at 680nm place is ordinate zou mapping, sees Fig. 5.The inverse of cut-off line slope is K value.Usual K value should within the scope of 95-100.As calculated, K=98, the requirement of conformance with standard curve.Survey enzyme work/OD ₆₀₀be unit cell enzymatic productivity, result unit cell enzymatic productivity recombinant bacterium comparatively wild mushroom improves about 278%.

(5) recombinant bacterium genetic stability checking

Do not gone down to posterity 100 times containing the 1/3LB flat board of resistance is rule continuously by genetic engineering bacterium, the bacterial classification getting for 10,20,30,40,50,60,70,80,90,100 generations respectively ferments, and fermentation condition is the same.Experimental result is that unit cell yield of enzyme keeps stable, and recombinant bacterium hereditary property is stablized.

Claims (1)

1. a bacillus engineering bacteria, comprise the linear fragment of sequence 8, described plasmid is using the sequence of 16Sr DNA as homology arm, be card that resistance screening marker gene, P43 promotor, multiple clone site, terminator sequence between homology arm, the linear fragment of described sequence 8 is connected to the pstI restriction enzyme site of plasmid pMD18Tsimple-16SrDNA, and concrete building process is as follows:

The structure of homologous recombination vector

[1]. the amplification of each gene

(1) the gene amplification of genus bacillus 16S rDNA

Be that template carries out PCR with Alkaliphilic bacillus, described Alkaliphilic bacillus through the homology of the whole bacillus 16SrDNA of sequence alignment more than 95%, therefore the genome of choosing a kind of bacterium is wherein as template, again by agarose gel electrophoresis and cut glue reclaim obtain goal gene fragment, required primer is as follows:

P1 5′-CGGATATC GAGAGTTTGATCCTGGCTGGCTCAG-3′

P2 5′-CGGATATC AAGGAGGTGATCCAGCCGCA-3′

Reaction system is 50 μ L, and proportioning is as follows:

PCR program is as follows:

95 DEG C of denaturation 5min; 94 DEG C of sex change 45s; 57 DEG C of annealing 45s; 72 DEG C extend 1min30s; 30 rear 72 DEG C of extension 10min of circulation; 4 DEG C of preservations,

(2) block the amplification of that resistant gene (kan)

With plasmid pBE2 for template carries out PCR, then by agarose gel electrophoresis and cut glue reclaim obtain goal gene fragment, required primer is as follows:

P3 5′-CGGACTAGTGCCGATGAAGATGGATTTTCTATTA-3′

P4 5′-GGGACTAGTGCGCCCCGGGTTCGAAGGGCA-3′

Reaction system is 50 μ L, and proportioning is as follows:

PCR program is as follows:

95 DEG C of denaturation 5min; 94 DEG C of sex change 45s; 58 DEG C of annealing 45s; 72 DEG C extend 1min30s; 30 rear 72 DEG C of extension 10min of circulation; 4 DEG C of preservations,

(3) the amplification of mature peptide gene apr4

First, with Alkaliphilic bacillus genome for template carries out PCR, then by agarose gel electrophoresis and cut glue reclaim obtain goal gene fragment, required primer is as follows:

P5 5′-CGCGGATCCCAATCAGTGCCATGGGGAAT-3′

P6 5′-CCCAAGCTTTTAGCGTGTTGCCGCTTC-3′

Reaction system is 50 μ L, and proportioning is as follows:

PCR program is as follows:

95 DEG C of denaturation 5min; 94 DEG C of sex change 45s; 58 DEG C of annealing 45s; 72 DEG C extend 1min30s; 30 rear 72 DEG C of extension 10min of circulation; 4 DEG C of preservations,

[2]. the structure of general recombinant vectors pKD314, building process is as follows

(1) after being connected with 16SrDNA gene by plasmid pMD18Tsimple, obtain plasmid pMD18Tsimple-16SrDNA,

(2) search P43 promoter sequence according to NCBI GenBank accession No.K02174.1 and GenBank accession No.AAA22212 searches terminator sequence, according to the sequence of the sequences Design synthesis of report, multiple clone site is introduced between promotor and terminator, concrete sequence psg is shown in sequence 7, sequent synthesis entrusts invitrogen company, the plasmid called after pUC57-psg of synthesis

(3) the SpeI restriction enzyme site linking pUC57-psg after being cut by that resistant gene of card kan restriction enzyme SpeI enzyme obtains plasmid pUC57-kan-psg,

(4) plasmid pUC57-kan-psg restriction enzyme pstI enzyme is cut and is obtained linear fragment, described linear fragment is shown in sequence 8, this linear fragment is connected on plasmid pMD18Tsimple-16SrDNA and obtains plasmid pKD314, wherein plasmid pMD18Tsimple-16SrDNA also needs to cut through pstI enzyme, and then connect

Above vector construction process ligase enzyme used is T4DNA ligase enzyme, and linked system is 20 μ L, and linked system is as follows: goal gene: the ratio=3:1-9:1 of the volumetric molar concentration of carrier, adds the mixing of appropriate 0.2ul ligase enzyme, at 16 DEG C, and reaction overnight,

The enzyme system of cutting is 50 μ L, and concrete proportioning is,

37 DEG C of enzymes cut 3h, add 10 × Loading Buffer 5 μ L termination reaction that restriction enzyme is subsidiary in digestion products, and 1% agarose gel electrophoresis detects digestion products,

[3] structure of high yield alkali protein Alkaliphilic bacillus

(1) the structure of recombinant plasmid pKD314-apr4M

PKD314 is connected with Sumizyme MP mature peptide gene apr4 after BamHI with HindIII double digestion, obtains recombinant plasmid pKD314-apr4,

Vector construction process ligase enzyme used is T4DNA ligase enzyme, and linked system is 20 μ L, and linked system is as follows: goal gene: the ratio=3:1-9:1 of the volumetric molar concentration of carrier, adds the mixing of appropriate 0.2ul ligase enzyme, at 16 DEG C, and reaction overnight,

ECORV double digestion recombinant plasmid obtains homologous recombination linear fragment, and the enzyme system of cutting is 50 μ L,

37 DEG C of enzymes cut 3h, add 10 × Loading Buffer 5 μ L termination reaction that restriction enzyme is subsidiary in digestion products, and 1% agarose gel electrophoresis detects digestion products, and to the linear fragment cutting glue recovery 4.3kb, plasmid PCR is verified;

(2) the electricity of recombinant fragment turns

After frozen Alkaliphilic bacillus competent cell TCCC11263 is dissolved on ice, get 50 μ L add restructuring linear fragment mix, then transfer in the electric shock cup of ice precooling, after using this cell mixture electricimpulse of high-voltage pulse electric shock conversion electric shock to terminate, rapidly cell suspension is transferred in the EP pipe of room temperature recovery medium, shaking culture for some time, get 150 μ L conversion fluids after the serial dilution of recovery wild Oryza species and be coated with 1% skim milk plate, kantlex containing 30 μ g/mL, screening positive clone;

(3) the checking of recombinant conversion

Select the single bacterium colony in resistant panel, be inoculated in containing in 40 μ g/mL kantlex LB substratum, 32 DEG C of shaking tables cultivate 24 hours, extract genome,

PCR verifies, checking primer is P1, P2,

Reaction system is 20 μ L, and proportioning is as follows:

PCR program is as follows:

95 DEG C of denaturation 5min; 94 DEG C of sex change 45s; 61 DEG C of annealing 45s; 58 DEG C extend 1min30s; 30 rear 72 DEG C of extension 10min of circulation; 4 DEG C of preservations.