CN1880461A - Escherichia coli self-cracking method and its dedicated carrier and application - Google Patents
Escherichia coli self-cracking method and its dedicated carrier and application Download PDFInfo
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Abstract
The invention discloses a colibacillus self-splitting method and specific carrier and appliance, which is characterized by the following: the carrier connects colibacillus expressing carrier with ultraviolet promoter, bacteriophage splitting gene and colibacillus terminal from 5' to 3'sequently; the colibacillus self-splitting carrier is conducted in the colibacillus to obtain recombinant colibacillus, which is shined by ultraviolet to split colibacillus cell. The experiment certifies that the splitting gene SRRz of Lambda bacteriophage can be expressed under ultraviolet induced condition.
Description
Technical field
The present invention relates to escherichia coli self-cracking method and dedicated carrier thereof and application, particularly relate to a kind of escherichia coli self-cracking method and dedicated carrier thereof and the application of this method in the high flux screening intracellular enzyme.
Background technology
Molecular orientation evolvement technology (Molecular Directed Evolution) develops (Frances H.Arnold by U.S. academician of the Chinese Academy of Engineering, the professor Frances Arnold of chemical industry system of Caltech (California Institute of Technology) in the initial stage nineties, Combinatorial and computationalchallenges for biocatalyst design.Nature.2001,409:253-257).This technology is the result of complementary development of genetically engineered, protein structure and computing technique and infiltration, indicates that the mankind can and need engineered protein (enzyme) according to own wish, even designs occurring in nature script non-existent brand-new protein (enzyme).
The molecular orientation evolvement technology, briefly be exactly to simulate Darwinian natural evolution principle, in breadboard test tube, will encode the gene of a certain protein (enzyme) with random mutation (random mutagenesis) and the method for hybridizing at random, recombinating to produce a large amount of sudden changes, make up the sudden change storehouse, (screening) screened in the mutation of these protein (enzyme) one by one or carry out the selection (selection) of " survival of the fittest " formula according to the specific function index then, thereby obtain the good mutation on specified property.With evolution is different naturally, the molecular orientation evolvement technology is to take place under artificial initiation conditions, and get rid of other direction sudden change by the evolution of selecting molecular group after the sudden change to keep a certain direction, whole evolutionary process by in advance specific, promptly is directed fully.
Molecular orientation evolvement technology (Frances H.Arnold, Combinatorial and computationalchallenges for biocatalyst design.Nature.2001,409:253-257; Frances H.Arnold.When blind is better:Protein design by evolution.NatureBiotechnology.1998,16:617-618; Hyun Joo, Zhanglin Lin, and Frances H.Arnold, Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation, Nature.1999,399:670-673) belong to a kind of " from bottom to top " irrational design (irrational design) (bottom-up) to protein (enzyme) molecule, avoided requirement to protein (enzyme) molecular structure and structure and functional dependency cognition, simply by simulation Darwin principle of evolving naturally, on molecular level, protein (enzyme) is carried out randomly changing, and in the progressive mode of multistep, from numerous gene mutation storehouses, obtain the mutation of improved properties by screening (or selection), make protein foreshorten to several years even some months in the evolutionary process that nature needs just can finish in millions of years, widened the research field and the range of application of protein (enzyme) engineering greatly, therefore illustrated that also the orthogenesis technology has very strong operability.
Because intestinal bacteria (Escherichia coli) genetic background is clear, easily cultivate, growth is quick, and has a large amount of alternative cloning vectors and expression vector, therefore selects for use intestinal bacteria very common as the exogenous protein expression system in the molecular orientation evolvement process.But because enzyme is demarcated in expression vector inside at first, and the substrate/product of enzyme is all external at expression vector, and most being difficult to enters cell through carrier cell, this high flux screening work of carrying out according to the activity of target protein (enzyme) in the evolution process of giving has brought great difficulty, therefore exploitation can be discharged into the recombinant protein (target protein) of biologically active outside the thalline or the simple and easy method in the substratum, will be significant to high flux screening.
At present, discharging the method for recombinant protein outside thalline mainly is by smudge cells (Anton P.J.Middelberg, Process-scale disruption of microorganisms, BiotechnologyAdvances.1995,13:491-551), comprise that mechanical means such as French press cracking, ultrasonication cracking (ultrasonication), vibration grind (vibration mills) cracking etc. and non-mechanical approach such as methods such as chemical reagent cracking, alkaline lysis and enzyme liberating.Mechanical means is difficult in the high flux screening and realizes, and non-mechanical approach need be added chemical reagent or enzyme usually, thereby brings interference problem for the mensuration of follow-up enzyme, and has increased the operation and the cost (as the use of a large amount of medical disposable materials) of high flux screening.
(Escherichia coli) is host's phage with intestinal bacteria, can be under certain external condition (as temperature, chemical substance, ultraviolet etc.) lysing cell, the product of accumulation in the cell is discharged.This method has broken wall efficient height, advantage such as controlled, easy to operate, for high flux screening provides possible means.The Lambda phage of intestinal bacteria (Escherichia coli) is that research at present is the clearest, one of most widely used phage.Cause the gene of lysis to comprise S, R and three genes of Rz in the Lambda phage, wherein the R genes encoding is a kind of water-soluble transglycosylase (transglycosylase), can cause the hydrolysis of peptide bond, peptidoglycan (the Corchero J.L. that decomposes cell walls, Rafael C.etc, Cell lysis in Escherichia colicultures stimulates growth and biosynthesis of recombinant proteins insurviving cells, Microbiol.Res.2001,156:13-18).The product of Rz gene may be a kind of endopeptidase (endopepidase), it can cut between peptidoglycan and the oligosaccharides and/or peptidoglycan and cell walls adventitia between be connected (Ry Young, Bacteriophage lysis:Mechanism and regulation, Microbiological Reviews.1992,56:430-381).The function of R and Rz gene product all is the degradation of cell wall, and S gene (Ronald R., Gregory N., Charles S., Jeffery S., Ry Young, Dominancein Lambda S mutations and evidence for translational control. J.Mol.Biol.1988,199:95-105; Chung Y.C., Kiebang N., Ry Young, S gene Expression andthe timing of lysis by bacteriophage, J.Bacteriol.1995,177:3283-3294) effect of product is the permeability that changes cytoplasmic membrane, on cytoplasmic membrane, form the porous structure,, and act on cell walls so that the product of R and Rz gene passes cytoplasmic membrane, make the cell walls fragmentation, discharge intracellular organic matter.
SOS repair system (Lewis L.K., Harlow G.R., Gregg-Jolly L.A., Mount DW., Identification of high affinity binding sites for LexA which define new DNAdamage-inducible genes in Escherichia coli.J.Mol.Biol.1994,241:507-23; Celina Janion, Some aspects of the SOS response system-A criticai survey, Acta Biochimica Polonica.2001,48:599-610), be to sustain damage (as uv irradiating) or dna replication dna when being subjected to hinder producing single stranded DNA as DNA, will cause that a series of SOS repairs the great expression of gene, and depending on RecA albumen, the great expression of these genes under the ultraviolet effect, activates, hydrolysis aporepressor LexA impels the reparation gene great expression in aporepressor downstream.Its principle as shown in Figure 1, wherein LexA is called SOS box with the calmodulin binding domain CaM of repairing upstream region of gene, this section encoding gene be positioned at repair gene promotor-35 to-10 zones.
Summary of the invention
The purpose of this invention is to provide a kind of escherichia coli self-cracking carrier that is used for the high flux screening intracellular enzyme.
Escherichia coli self-cracking carrier provided by the present invention is to be connected with ultraviolet promotor, the coli expression carrier of phage splitting gene and intestinal bacteria terminator in turn from 5 ' to 3 ' end.
Described ultraviolet promotor can be any one uv induction promotor, is preferably recA promotor or umuDC promotor; Described recA promotor has the nucleotide sequence of sequence 1 in the sequence table, and the umuDC promotor has the nucleotide sequence of sequence 2 in the sequence table.
Described phage splitting gene can be any one phage splitting gene, is preferably the lysis genes SRRz of Lambda phage, the nucleotide sequence with sequence 3 in the sequence table.
Described intestinal bacteria terminator also can be any one intestinal bacteria terminator, is preferably the rrnB terminator, has the nucleotide sequence of sequence 4 in the sequence table.
The carrier that sets out that is used to make up described carrier can be any one coli expression carrier, as pUC18, pUC19 or pET30a etc.
Be the carrier that sets out with pUC18, the escherichia coli self-cracking carrier of structure is pUC18-recA-SRRz-rrnB or pUC18-umuDC-SRRz-rrnB.
The recombination bacillus coli that contains described escherichia coli self-cracking carrier also belongs to protection scope of the present invention.
Second purpose of the present invention provides a kind of colibacillary autothermic cracking method.
Colibacillary autothermic cracking method provided by the present invention is that above-mentioned escherichia coli self-cracking carrier is imported in the intestinal bacteria, obtains recombination bacillus coli, recombination bacillus coli is shone the Bacillus coli cells cracking under ultraviolet ray again.
Described ultraviolet induction intensity is 3000-5000J/cm
2S, irradiation time is 30s-4min.
Above-mentioned escherichia coli self-cracking method is applicable to any intestinal bacteria.
The 3rd purpose of the present invention provides a kind of expression screening method of intracellular enzyme.
The expression screening method of intracellular enzyme provided by the present invention may further comprise the steps:
1) foreign gene is inserted in the above-mentioned escherichia coli self-cracking carrier, obtains intracellular enzyme expression screening carrier;
2) the intracellular enzyme expression screening carrier that contains foreign gene that step 1) is made up imports intestinal bacteria, obtains recombination bacillus coli;
3) fermentation recombination bacillus coli adds inductor intracellular enzyme is expressed, then recombination bacillus coli shone under ultraviolet ray, and the Bacillus coli cells cracking, intracellular enzyme is discharged in the fermented liquid;
4) intracellular enzyme in the fermented liquid is screened, obtain target protein.
In above-mentioned expression screening method, step 2) intestinal bacteria in can be the coli strain that is suitable for protein expression arbitrarily, as E.coli BL21, BL21 (DE3), HB101, XL1-Blue or DH5-α etc.
For improving transformation efficiency, the intracellular enzyme expression screening carrier that contains foreign gene that step 1) is made up imports colibacillary method and is preferably the electroporation conversion method, but also can adopt method commonly used in other bioengineering field, for example, calcium chloride transformation, lithium chloride conversion method, protoplast transformation method etc.
Step 3) is selected suitable inductor according to concrete used carrier and host bacterium, as for pUC18 or pET30a being the escherichia coli self-cracking carrier of vector construction of setting out, (Isopropyl-β-D-thigalactopyranoside is IPTG) as inductor can to adopt isopropyl ss-D thiogalactoside.Can carry out determination of activity to the intracellular enzyme in the fermented liquid according to ordinary method in the step 4), obtain active high target protein through screening.
The invention provides a kind of colibacillary autothermic cracking method and dedicated carrier thereof.Experimental results show that this carrier can make the lysis genes SRRz of Lambda phage obtain to express under uv induction, thereby make the host cell cracking, the external source target protein that discharges biologically active arrives substratum, is beneficial to the carrying out of high flux screening follow-up in the orthogenesis; Enzyme biopsy through the reporter protein beta-galactosidase enzymes is surveyed, and lysis efficiency can reach 63.7%.In addition, under the uv induction condition high flux screening that available carrier of the present invention carries out intracellular enzyme, have cheapness, advantage efficiently.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is the schematic diagram of SOS repair system
Fig. 2 is the structure synoptic diagram of pUC18-recA-SRRz-rrnB and pUC18-umuDC-SRRz-rrnB plasmid
Fig. 3 is synthetic recA promoter sequence figure
Fig. 4 is synthetic umuDC promoter sequence figure
Fig. 5 is expression and the lysis efficiency detected result of lysis genes SRRz under uv induction promotor recA control
Fig. 6 is expression and the lysis efficiency detected result of lysis genes SRRz under uv induction promotor umuDC control
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and all primers, gene order are synthesized and examining order is finished by the living worker in Shanghai.
The structure of embodiment 1, escherichia coli self-cracking carrier
Now be the carrier that sets out with pUC18 (TaKaRa company), make up intracellular enzyme expression screening dedicated carrier pUC18-recA-SRRz-rrnB and pUC18-umuDC-SRRz-rrnB, the synoptic diagram of plasmid construction is referring to Fig. 2, and concrete grammar is as follows:
One, the structure of pUC18-recA-SRRz-rrnB
1, makes up recombinant vectors pUC18-recA-rrnB
1) synthetic recA promotor
Nucleotide sequence (sequence 1 in the sequence table) synthetic recA promoter sequence according to the recA promotor, and at its 5 ' end interpolation restriction enzyme Sap I recognition site, 3 ' end adds restriction enzyme Xho I and EcoR I recognition site, and sequence constitutes as shown in Figure 3.
2) pcr amplification rrnB terminator sequence
Nucleotide sequence (sequence 4 in the sequence table) design pcr amplification primer according to the rrnB terminator, and add restriction enzyme EcoR I and Nco I recognition site at 5 ' end of upstream primer, 5 ' end of downstream primer adds restriction enzyme A fl III recognition site, and primer sequence is as follows:
P1 (forward primer):
P2 (reverse primer):
Extract the genomic dna of E.coli DH5-α and as template, under the guiding of primer P1 and P2, pcr amplification rrnB terminator sequence.
3) reorganization
With restriction enzyme Sap I and Afl III plasmid pUC18 is carried out double digestion; RrnB terminator to pcr amplification carries out double digestion with restriction enzyme EcoR I and Afl III; Two kinds of double digestion products are connected with synthetic recA promotor, to connect product Transformed E .coli BL21 competent cell, transformant is inoculated in LB resistance culture plate, and (every liter of substratum contains Tryptones 10.0g, yeast extract powder 5.0g, NaCl 10.0g, agar 15.0g, pH7.0, penbritin (Ampicillin) 50 μ g/mL) screening positive clone, the upgrading grain, obtain being connected with the recombinant plasmid of recA promotor and rrnB terminator, called after pUC18-recA-rrnB.
2, pcr amplification Lambda phage SRRz lysis genes
Nucleotide sequence (sequence 3 in the sequence table) design pcr amplification primer according to the SRRz lysis genes, and add restriction enzyme Xho I recognition site at 5 ' end of upstream primer, 5 ' end of downstream primer adds restriction enzyme Nco I recognition site, and primer sequence is as follows:
P3 (upstream primer):
P4 (downstream primer):
Extract Lambda phage (Lambda DNA, Sam7) genomic dna and as template, under the guiding of primer P3 and P4, pcr amplification SRRz lysis genes, and add restriction enzyme XhoI and Nco I recognition site respectively at the gene two ends (because the SRRz gene of Lambda phage is " S " disappearance, terminator codon in " S " gene that now uses the method for rite-directed mutagenesis to remove, thereby obtain to have the SRRz lysis genes of complete function).
3, the acquisition of pUC18-recA-SRRz-rrnB
SRRz gene to reorganization plasmid pUC18-recA-rrnB and pcr amplification carries out double digestion with restriction enzyme Xho I and Nco I, connect, to connect product Transformed E .coli BL21 competent cell again, transformant is inoculated in the LB resistance culture plate screening positive clone identical with step 1, the upgrading grain, obtain being connected with the recombinant plasmid of recA promotor, SRRz and rrnB terminator, called after pUC18-recA-SRRz-rrnB.
Two, the structure of pUC18-umuDC-SRRz-rrnB
1, the umuDC promotor is synthetic
Nucleotide sequence (sequence 2 in the sequence table) synthetic umuDC promoter sequence according to the umuDC promotor, and at its 5 ' end interpolation restriction enzyme Sap I recognition site, 3 ' end adds restriction enzyme Xho I recognition site, and sequence constitutes as shown in Figure 4.
2, the acquisition of pUC18-umuDC-SRRz-rrnB
UmuDC promotor to reorganization plasmid pUC18-recA-rrnB and pcr amplification is carried out double digestion with restriction enzyme Sap I and Xho I, connect, to connect product Transformed E .coli BL21 competent cell again, transformant is inoculated in the LB resistance culture plate screening positive clone identical with step 1, the upgrading grain, obtain being connected with the recombinant plasmid of umuDC promotor, SRRz and rrnB terminator, called after pUC18-umuDC-SRRz-rrnB.
Embodiment 2, (β-galactosidase) is as the validity check of the SRRz lysis genes of reporter protein with beta-galactosidase enzymes
Select the positive colony that carries escherichia coli self-cracking plasmid vector pUC18-recA-SRRz-rrnB and pUC18-umuDC-SRRz-rrnB among the embodiment 1 respectively, be inoculated in respectively in the LB liquid nutrient medium, shake-flask culture is to OD under 30 ℃, 250rpm
600Value is induced the expression of reporter protein beta-galactosidase enzymes with the IPTG of final concentration 1mM when the 0.4-0.5, and inducing culture 1 hour is to OD
600Value is 0.8-1.0, is 3840J/cm in ultraviolet ray (UV) intensity again
2(6mL bacterium liquid/culture dish) induces 40s under the s, continue under the same conditions then to cultivate 2,4,6 hours and (18-20 hour) cultivation back harvested cell that spends the night are measured the inside and outside beta-galactosidase enzymes enzyme of cell (Miller J.H. alive, Experiments in Molecular Genetics, 352-355, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1972)), calculate the lysis efficiency of each time point simultaneously.Each sample is parallel to be done 3 times, sets up control group (non-irradiated with ultraviolet radiation) simultaneously.
Wherein, the extracting method of intracellular enzyme (beta-galactosidase enzymes) may further comprise the steps:
1) behind uv induction, cultivates 0,2,4 and spend the night (18-20 hour) after, gather in the crops the bacterium liquid of 0.5mL respectively, the centrifugal 5min of 13000rpm gets supernatant and surveys the extracellular enzyme activity (activity determination method can reference: Sambrook J., Russell, D.W.Molecular cloning:A laboratory Manual, 3
RdEd.Cold SpringHarbor Laboratory Press, New York (2001));
2) add 0.5mL bacterium lysis buffer (50mM pH 7.2 Tris-Cl, 5% glycerine, 50mM NaCl) re-suspended cell;
3) re-suspended cell liquid is placed the freezing 1min of liquid nitrogen, take out to be placed in 25 ℃ of water-baths and melt, so multigelation is 3 times;
4) ultrasonication in 1.5mL EP pipe;
5) adding N,O-Diacetylmuramidase to final concentration in suspension is 0.2mg/mL, and room temperature was shaken (100rpm) 1 hour gently on decolorization swinging table;
6) 13, the centrifugal 4min of 000rpm gets supernatant and carries out enzyme assay, and the enzyme work of surveying is intracellular enzyme and lives.
Detection method of betagalactosidase activity (microplate reader) and lysis efficiency method of calculation are as follows:
The activity of beta-galactosidase enzymes is to measure by chromogenic substrate ortho-nitrophenyl-β-D-galactopyranoside (oNPG is available from Sigma company), and product has maximum absorption band at the 420nm place, therefore by measuring the absorbance A at 420nm place
420, use following formula: enzyme=1000 * dilution factor alive * A
420/ (t * V * OD
600), wherein t is illustrated in the reaction times under 28 ℃, V represents the volume of harvested cell, can calculate in the born of the same parents, extracellular enzyme is lived (MillerJ.H., Experiments in Molecular Genetics, 352-355, Cold Spring HarborLaboratory, Cold Spring Harbor, New York (1972)).Then beta-galactosidase enzymes in the born of the same parents that extracted, outside the born of the same parents is measured enzyme kineticss with microplate reader SpectroMAX 190 in 96 orifice plates, to obtain A
420/ t.Wherein, for guaranteeing measured absorbancy in linearity range, the method with stepwise dilution obtains linear extension rate earlier.In 96 orifice plates, the reaction system in every hole is: 125 μ l enzyme liquid (needing dilution in case of necessity, the phosphate buffered saline buffer of pH7.6) add 50 μ l 4g/L oNPG.The kinetic determination of 30min in the timed interval of 2min, obtains absorbance A
420Rate can calculate enzyme and live over time.
The lysis efficiency of thalline can calculate by this formula: lysis efficiency=extracellular enzyme work/born of the same parents are interior, the outer total enzyme of born of the same parents is lived.
The result is under the control of uv induction promotor recA, and Lambda phage splitting gene SRRz obtains to express, and makes the effective cracking of thalline.Behind the uv induction 40s, the cracking of reorganization thalline part has some intracellular organic matters to be discharged in the substratum, and the concentration (OD of thalline
600) along with the increase of incubation time descend gradually (see among Fig. 5 figure a).At this moment, determination of activity by reporter protein-beta-galactosidase enzymes, calculating lysis efficiency is 63.7% (seeing the figure b among Fig. 5), and this high flux screening to recombinant protein is enough, but further growth along with thalline, as behind the uv induction 4 hours, after 6 hours and the incubated overnight (18-20) hour, remaining uncracked thalline is grown again, the calculating born of the same parents are outer, intracellular enzyme is lived and lysis efficiency, the result all has in various degree and to reduce, particularly cultivate 18-20 hour after, lysis efficiency only is 27.4%.Control group is not having under the condition of uv induction, and thalline does not have cracking, and SRRz does not almost have background to express, and shows that the strictness of recA promotor is regulated and control by ultraviolet.
Under the control of uv induction promotor umuDC, cracking process and lysis efficiency are very close with situation under recA regulation and control, and the thalline background cracking level of umuDC regulation and control that different is is a little more than latter's (the recA regulation and control down) (seeing figure a and figure b among Fig. 6).
Above-mentioned detected result shows with escherichia coli self-cracking carrier of the present invention can obtain cellular lysate effect preferably, can be used for the high flux screening of recombinant protein.
Sequence table
<160>4
<210>1
<211>35
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
gctacttgat?actgtatgag?catacagtat?aattg 35
<210>2
<211>50
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
gcttattgac?atgctggcaa?gaacagacta?ctgtatataa?aaacagtata 50
<210>3
<211>1549
<212>DNA
＜213〉Lambda phage
<400>3
gccactgtct?gtcctgaatt?cattagtaat?agttacgctg?cggcctttta?cacatgacct 60
tcgtgaaagc?gggtggcagg?aggtcgcgct?aacaacctcc?tgccgttttg?cccgtgcata 120
tcggtcacga?acaaatctga?ttactaaaca?cagtagcctg?gatttgttct?atcagtaatc 180
gaccttattc?ctaattaaat?agagcaaatc?cccttattgg?gggtaagaca?tgaagatgcc 240
agaaaaacat?gacctgttgg?ccgccattct?cgcggcaaag?gaacaaggca?tcggggcaat 300
ccttgcgttt?gcaatggcgt?accttcgcgg?cagatataat?ggcggtgcgt?ttacaaaaac 360
agtaatcgac?gcaacgatgt?gcgccattat?cgcctggttc?attcgtgacc?ttctcgactt 420
cgccggacta?agtagcaatc?tcgcttatat?aacgagcgtg?tttatcggct?acatcggtac 480
tgactcgatt?ggttcgctta?tcaaacgctt?cgctgctaaa?aaagccggag?tagaagatgg 540
tagaaatcaa?taatcaacgt?aaggcgttcc?tcgatatgct?ggcgtggtcg?gagggaactg 600
ataacggacg?tcagaaaacc?agaaatcatg?gttatgacgt?cattgtaggc?ggagagctat 660
ttactgatta?ctccgatcac?cctcgcaaac?ttgtcacgct?aaacccaaaa?ctcaaatcaa 720
caggcgccgg?acgctaccag?cttctttccc?gttggtggga?tgcctaccgc?aagcagcttg 780
gcctgaaaga?cttctctccg?aaaagtcagg?acgctgtggc?attgcagcag?attaaggagc 840
gtggcgcttt?acctatgatt?gatcgtggtg?atatccgtca?ggcaatcgac?cgttgcagca 900
atatctgggc?ttcactgccg?ggcgctggtt?atggtcagtt?cgagcataag?gctgacagcc 960
tgattgcaaa?attcaaagaa?gcgggcggaa?cggtcagaga?gattgatgta?tgagcagagt 1020
caccgcgatt?atctccgctc?tggttatctg?catcatcgtc?tgcctgtcat?gggctgttaa 1080
tcattaccgt?gataacgcca?ttacctacaa?agcccagcgc?gacaaaaatg?ccagagaact 1140
gaagctggcg?aacgcggcaa?ttactgacat?gcagatgcgt?cagcgtgatg?ttgctgcgct 1200
cgatgcaaaa?tacacgaagg?agttagctga?tgctaaagct?gaaaatgatg?ctctgcgtga 1260
tgatgttgcc?gctggtcgtc?gtcggttgca?catcaaagca?gtctgtcagt?cagtgcgtga 1320
agccaccacc?gcctccggcg?tggataatgc?agcctccccc?cgactggcag?acaccgctga 1380
acgggattat?ttcaccctca?gagagaggct?gatcactatg?caaaaacaac?tggaaggaac 1440
ccagaagtat?attaatgagc?agtgcagata?gagttgccca?tatcgatggg?caactcatgc 1500
aattattgtg?agcaatacac?acgcgcttcc?agcggagtat?aaatgccta 1549
<210>4
<211>246
<212>DNA
＜213〉intestinal bacteria (Escherichia coli)
<400>4
aggcatcaaa?taaaacgaaa?ggctcagtcg?aaagactggg?cctttcgttt?tatctgttgt 60
ttgtcggtga?acgctctccg?agtaggacaa?atccgccggg?agcggatttg?aacgttgcga 120
agcaacggcc?cggagggtgg?cgggcaggac?gcccgccata?aactgccagg?catcaaatta 180
agcagaaggc?catcctgacg?gatggccttt?ttgcgtttct?acaaactctt?cctgtcgtca 240
tatcta 246
Claims (12)
1, escherichia coli self-cracking carrier is to be connected with ultraviolet promotor, the coli expression carrier of phage splitting gene and intestinal bacteria terminator in turn from 5 ' to 3 ' end.
2, carrier according to claim 1 is characterized in that: described ultraviolet promotor is recA promotor or umuDC promotor; Described recA promotor has the nucleotide sequence of sequence 1 in the sequence table, and the umuDC promotor has the nucleotide sequence of sequence 2 in the sequence table.
3, carrier according to claim 1 is characterized in that: the lysis genes SRRz that described phage splitting gene is the Lambda phage, the nucleotide sequence with sequence 3 in the sequence table.
4, carrier according to claim 1 is characterized in that: described terminator is the rrnB terminator, has the nucleotide sequence of sequence 4 in the sequence table.
5, according to the arbitrary described carrier of claim 1-4, it is characterized in that: the carrier that sets out that is used to make up described carrier is pUC18, pUC19 or pET30a.
6, carrier according to claim 5 is characterized in that: described escherichia coli self-cracking carrier is pUC18-recA-SRRz-rrnB or pUC18-umuDC-SRRz-rrnB.
7, the recombination bacillus coli that contains the described carrier of claim 1.
8, a kind of escherichia coli self-cracking method is that the described escherichia coli self-cracking carrier of claim 1 is imported in the intestinal bacteria, obtains recombination bacillus coli, recombination bacillus coli is shone the Bacillus coli cells cracking under ultraviolet ray again.
9, method according to claim 8 is characterized in that: described uitraviolet intensity is 3000-5000J/cm
2S, irradiation time is 40s-4min.
10, the application of the described escherichia coli self-cracking method of claim 8 in the intracellular enzyme expression screening.
11, application according to claim 10 is characterized in that: the expression screening method of described intracellular enzyme may further comprise the steps:
1) foreign gene is inserted in the described escherichia coli self-cracking carrier of claim 1, obtains intracellular enzyme expression screening carrier;
2) the intracellular enzyme expression screening carrier that contains foreign gene that step 1) is made up imports intestinal bacteria, obtains recombination bacillus coli;
3) fermentation recombination bacillus coli adds inductor intracellular enzyme is expressed, then recombination bacillus coli shone under ultraviolet ray, and the Bacillus coli cells cracking, intracellular enzyme is discharged in the fermented liquid;
4) intracellular enzyme in the fermented liquid is screened, obtain target protein.
12, application according to claim 11 is characterized in that: the intestinal bacteria described step 2) are E.coli BL21, BL21 (DE3), HB101, XL1-Blue or DH5-α.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636164A (en) * | 2017-01-18 | 2017-05-10 | 华南理工大学 | Genetic toxicant detection vector and detection method |
| CN106906209A (en) * | 2017-03-09 | 2017-06-30 | 华南理工大学 | A kind of DNA damage detects response element and its application |
| CN110873790A (en) * | 2018-09-03 | 2020-03-10 | 华南理工大学 | Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof |
| CN112680498A (en) * | 2020-12-28 | 2021-04-20 | 华南理工大学 | High-throughput screening method for genotoxic substances |
-
2006
- 2006-04-28 CN CNB2006100789520A patent/CN100448999C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636164A (en) * | 2017-01-18 | 2017-05-10 | 华南理工大学 | Genetic toxicant detection vector and detection method |
| WO2018133513A1 (en) * | 2017-01-18 | 2018-07-26 | 华南理工大学 | Genotoxic substance detection vector and detection method thereof |
| AU2017393714B2 (en) * | 2017-01-18 | 2021-08-05 | South China University Of Technology | Genotoxic substance detection vector and detection method thereof |
| CN106906209A (en) * | 2017-03-09 | 2017-06-30 | 华南理工大学 | A kind of DNA damage detects response element and its application |
| CN110873790A (en) * | 2018-09-03 | 2020-03-10 | 华南理工大学 | Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof |
| CN112680498A (en) * | 2020-12-28 | 2021-04-20 | 华南理工大学 | High-throughput screening method for genotoxic substances |
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