CN102080097A - Electroporation genetic manipulation method of Zymomonas mobilis - Google Patents
Electroporation genetic manipulation method of Zymomonas mobilis Download PDFInfo
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Abstract
The invention discloses an electroporation genetic manipulation method of Zymomonas mobilis, which comprises the following steps: carrying out electroporation to convert DNA (deoxyribonucleic acid) to be converted into competent cells of Zymomonas mobilis, or carrying out incubation on cell-free extract of Zymomonas mobilis and DNA to be converted, and carrying out electroporation to convert the incubated DNA into competent cells of Zymomonas mobilis. In the invention, a cell-free culture liquid of Zymomonas mobilis strains is used for treating the DNA to be converted so as to modify the DNA, thereby enhancing the conversion efficiency; the method disclosed by the invention can be used for carrying out electroporation on different hosts and DNAs by optimizing the key electroporation physical parameters, so as to enhance the conversion efficiency and stability of electroporation genetic manipulation of Zymomonas mobilis and adapt to all the genetic manipulations which are carried out through electroporation conversion and have different construction purposes, so that the method is also universal.
Description
Technical field
The invention belongs to the microbiological genetic engineering field.The electroporation genetic manipulation method that relates to a kind of zymomonas mobilis particularly.
Background technology
Current outstanding global energy crisis and problem of environmental pollution; make and utilize biotechnology and renewable resources (biomass) to carry out the suitability for industrialized production of alcohol fuel, become one of great development project of the many countries that comprise the U.S., Brazil, China etc.These countries all attempt exploitation new can be used for more direct, efficiently utilize the fuel ethanol production bacterial classification and the technology of biomass resource more comprehensively.And zymomonas mobilis ethanol fermentation speed is fast, alcohol yied is high (is 97% of theoretical value, and yeast is the 90-92% of theoretical value), can tolerate high glucose concentration and high determining alcohol, be to produce one of the strongest bacterium of pure ability known at present, becomes the alcohol production bacterial classification of great exploitation potential for its; Biochemical industry products such as zymomonas mobilis can also the high yield Polylevulosan, gluconic acid, sorbyl alcohol have industrial utilization and are worth.
But the carbon source scope that zymomonas mobilis can utilize is too narrow, only limits to glucose, fructose and sucrose.Expand the retrofit work of its substrate scope and obtained remarkable progress, in October, 2006, Dupont E.I.Du Pont Company and Broin company united the technology of having announced to produce based on the agricultural crop straw of zymomonas mobilis xylose utilization engineering strain alcohol, and had successively set up biomass alcohol production demonstration plant and pilot plant.Bacterial strain ZM4 (=ATCC31821) and the whole genome sequence of NCIMB11163 also respectively at delivering announcement (Seo JS in 2005,2009, Chong H, Park HS, et al.The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4.Nature Biotechnology.2005; 23:63-68.Vassili N.Kouvelis, Elizabeth Saunders, Thomas S.Brettin, et al.Complete Genome Sequence of the Ethanol Producer Zymomonas mobilis NCIMB 11163.Journal of Bacteriology, 2009,191 (22): 7140-7141.).Yet basic scientific research of zymomonas mobilis and genetic modification means present situation also can not satisfy the needs of applied research far away.The relative shortage of zymomonas mobilis genetic background knowledge, strict restriction modification system, extensive and various endogenous plasmid, resistance spectrum or the like factor widely make still limited and immature so far to its genetic manipulation means.
Import at present foreign gene and carry out genomic dna and modify (or chromosomal inheritance operation) and undertaken by conjugal transfer and conversion (chemical method, electroporation).Conjugal transfer work was just carried out as far back as the 1980s, but conjugal transfer efficient is very big because of bacterial strain and plasmid difference, is low generally.Chemical method transforms report seldom, and is low and be difficult to that repetition is few to be used because of efficient.Electroporation method is then convenient, simple, relatively stable, thereby uses extensive relatively.Some wide host range plasmid carriers are transformed in the different zymomonas mobilis bacterial strains by electroporation with the artificial intestinal bacteria-zymomonas mobilis shuttle vector that makes up, and can stablize and duplicate existence.These are all not directly related with the electroporation transformation efficiency of the plasmid vector of goal gene and host strain, plasmid type and concrete operational condition, method, and the transformation efficiency of report is from 10
1Individual/μ g DNA to 10
6Individual/μ g DNA does not wait, and analyzes its operational condition, gimmick, and then difference is very big again each other.
In general, plasmid is big more, and transformation efficiency is low more.Have the conversion of the reproducible plasmid of goal gene, more aforementioned much more difficult, disclosed information is much less also not with the conversion of the reproducible plasmid of goal gene, and the data report of discrete transformation efficiency is only arranged at present.
To zymomonas mobilis carry out genomic dna when modifying (or chromosomal inheritance operation) conventional adopt based on host self recA system, homologous recombination site-directed integration mode by long homology arm, or the mode of swivel base at random of being undertaken by the transposon element, all at first need to be correlated with plasmid or dna molecular, through electroporation and (or) mode of conjugal transfer, outside born of the same parents, be transferred in the born of the same parents; And plasmid or dna molecular enter integration, swivel base process behind the cell, all belong to rare event, so genomic dna modifies (or chromosomal inheritance operation), objectively more need electroporation method for transformation efficient, stable, good reproducibility.And the information of this respect is very limited especially at present.
In sum, present electroporation method for transformation, it all is special applications at specific bacterial strain and certain plasmid, be difficult to be generalized to other bacterial strain and plasmid, lack versatility and stability, repeated relatively, the needs that are difficult to satisfy the zymomonas mobilis molecular biology research and further transform applied research, more general, efficient, stable electroporation method need be set up in this area.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of rapid Optimum is provided, adapts to wide, stable, the electroporation genetic manipulation method of zymomonas mobilis efficiently.
Technical scheme of the present invention is summarized as follows:
The electroporation genetic manipulation method of a kind of zymomonas mobilis, comprise the steps: DNA to be transformed is transformed into by electroporation in the competent cell of zymomonas mobilis or with zymomonas mobilis cell-free extract and DNA to be transformed and hatch jointly, the DNA after will hatching by electroporation is transformed in the competent cell of zymomonas mobilis; The competent cell of described zymomonas mobilis is that zymomonas mobilis is cultured to OD
600nm=0.375～0.420 bacterium liquid concentrates 100 times and make; The parameter of electroporation operation is: the competent cell volume is 80 μ l～160 μ l in the 2mm electric shock cup; Strength of electric field is 11.0～14.0kV/cm; The recovery time is 3h～24h.
Described zymomonas mobilis cell-free extract is to make with following method: cultivate zymomonas mobilis to OD
600=0.6～1.0 nutrient solution, at 8000～12000rpm, 4 ℃, the centrifugal collection thalline of 3～6min, extract damping fluid HND washing with isopyknic ice bath precooling, extract damping fluid HND with the ice bath precooling of 1/20 to 1/10 volume of nutrient solution volume is resuspended, ultrasonic on ice broken wall, 14000～18000rpm, 4 ℃, 8～12min are centrifugal, collect supernatant liquor, described extract damping fluid HND is: the 200mM HEPES of pH=7.0 (N-(2-hydroxyethyl) piperazine-N '-2 sulfonic acid), 20mM Na
2EDTA, 5mM DTT (dithiothreitol (DTT)).
Described with zymomonas mobilis cell-free extract and DNA to be transformed hatch jointly for: select 100 μ l reaction systems, comprise 100mM HEPES, pH=7.0,10mM Na
2EDTA, 2.5mM DTT, 5mM MgCl
2, 1 μ M SAM (S-adenosylmethionine), 2～10 μ g DNA to be transformed, 50 μ l zymomonas mobilis cell-free extracts, 37 ℃, reaction 2～3 hours, purifying, sterilized water dissolving.
Described zymomonas mobilis is ZM4, CP4, ATCC10988 or ZM4 (ldh::FRT-cml-FRT).
The competent cell volume is 120 μ l in the described 2mm electric shock cup.
Described strength of electric field is 11.75～13.25kV/cm.
Beneficial effect of the present invention is:
1, utilize the acellular nutrient solution of zymomonas mobilis bacterial strain to handle DNA to be transformed, it to be modified, thereby the restriction of the host's restriction modification system when reducing electroporation and transforming improves transformation efficiency;
2, select plasmid vector according to different genetic manipulation types and purpose, select the higher plasmid of transformation efficiency to be the material construction recombinant plasmid that sets out;
3, utilize the crucial electroporation physical parameter of having optimized of the present invention that different hosts and DNA carried out electroporation, the transformation efficiency and the stability of zymomonas mobilis electroporation genetic manipulation have been improved, adapted to all and transformed genetic manipulations that make up purposes that carry out, different by electroporation, from and have a versatility.
Foreign gene conversion and genomic dna modification can be carried out by method of the present invention, thereby fundamental research and relevant applied research that zymomonas mobilis comprises gene function, expression regulation research can be widely used in.
Description of drawings
Fig. 1. 1-1 is shuttle vectors pZB21 (5930bp) among the figure, 1-2 is shuttle vectors pZB21-Plac-lacZ (6375bp), 1-3 is wide host's carrier pBBR1MCS-2 (5144bp), and 1-4 is the physical map of fixed point integration carrier pBR328-ldhR-cml-ldhL (7447bp).Wherein, Ec ori is the escherichia coli plasmid replicon, from plasmid pBR328; Zm ori is the zymomonas mobilis plasmid replicon, from plasmid pZM2; Tet is a tetracycline resistance gene, cml is a chloramphenicol resistance gene, amp is an ampicillin resistance gene, and Plac is the promotor of intestinal bacteria lac gene, and lacZ is a beta-galactosidase gene, kan is the kalamycin resistant gene, mob plasmid migration genes involved, rep plasmid replication genes involved, MCS are multiple clone site, ldh is a lactate dehydrogenase gene, and ldhR and ldhL are used separately as the homology arm of homologous recombination.
Fig. 2. the structure synoptic diagram of plasmid pBBR1MCS-2-Pgap-FLP (6880bp).
Fig. 3. electroporation transforms pBBR1MCS-2 (5144bp) to zymomonas mobilis bacterial strain ZM4 under the different cell concentration multiples
Fig. 4. electroporation transforms pZB21 (5930bp) to zymomonas mobilis bacterial strain CP4 under the different cell concentration multiples
Fig. 5. in the electric shock cup under the different feeling attitude cell volume electroporation transform pBBR1MCS-2 (5144bp) to zymomonas mobilis bacterial strain ZM4
Fig. 6. electroporation conversion pBBR1MCS-2 (5144bp), pZB21 (5930bp) and pBBR1MCS-2-Pgap-FLP (6880bp) are to ZM4 under the different strength of electric field
Fig. 7. electroporation conversion pBBR1MCS-2 (5144bp), pZB21 (5930bp) and pZB21-Plac-lacZ (6375bp) are to CP4 under the different strength of electric field
Fig. 8. electroporation transforms pBBR1MCS (5144bp), pZB21 (5930bp) to ZM4 under the different recovery times
Fig. 9. electroporation conversion pBBR1MCS-2 (5144bp), pZB21 (5930bp) and pZB21-Plac-lacZ (6375bp) are to CP4 under the different recovery times
Embodiment
In general, the three big restraining factors that influence the electroporation transformation efficiency are plasmid characteristic, host self restriction modification system and electroporation physical parameter.At these restraining factors, the present invention has taked relative counter measures respectively, specifically: 1, before electroporation, handle DNA to be transformed with the zymomonas mobilis cell-free extract, make DNA to be transformed obtain the modification of host's restriction modification system, overcome the host allogeneic dna sequence DNA is entered later restriction of host and Degradation; 2, select the higher plasmid of transformation efficiency for the material construction recombinant plasmid that sets out, transform again; 3, select crucial electroporation physical parameter to be optimized,, select the combination of different parameter optimization levels different hosts and DNA; These crucial electroporation physical parameters comprise: be used to prepare the density of the competent cell of the growth phase of bacterium liquid of electroporation competent cell and preparation, and competent cell volume in the electric shock cup, DNA concentration, strength of electric field adds the recovery time behind the resuscitation fluid.Because that the present invention to the integrated use of these measures, therefore provides is a kind of rapid Optimum, general, stable, method for transformation efficiently.
Should be understood that, because it is various to be used for the DNA type of electroporation conversion, its each other difference greatly (mainly refer to big or small, have or not replicon, copy type, the selective marker type, or the like), add bioprocess type difference in its related cell, the optimum operation condition of electroporation generally need be groped and be optimized, the method for a kind of rapid Optimum operational condition when technology of the present invention provides at new bacterial strain and new DNA type, thereby also had versatility, stability simultaneously.
Should be understood that, owing to the same reason, electroporation operation at different strains and different DNA types, its transformation efficiency varies, of the present invention efficient, not the height of simple transformation efficiency numerical value, but with respect to the common transformation efficiency of specific bacterial strain and specific DNA type (bioprocess in the specific cell is correspondingly arranged); Of the present invention efficient, for, the conversion that is difficult to realize very low, also can be understood as on the basis of rapid Optimum and realized conversion for those efficient own, promptly obtained the purpose transformant.
The plasmid type that is used for the conversion of zymomonas mobilis foreign gene that the present invention is selected has:
1, reproducible shuttle plasmid, comprise: 1) pZB21 (5930bp), the collection of illustrative plates of pZB21 is seen accompanying drawing 1, the structure of pZB21 is referring to document (Zou Shaolan, Gao Weihua, Liu Cheng etc. the structure of shuttle vectors between zymomonas mobilis and intestinal bacteria. Nankai University's journal (natural science edition), 2006,39 (3): 23-28); 2) pZB21-Plac-lacZ (6375bp), the collection of illustrative plates of pZB21-Plac-lacZ is seen accompanying drawing 1, and the structure of pZB21-Plac-lacZ is referring to document (Zhang Yiting, the research of homologous recombination method and abduction delivering system among the Zmobilis, University Of Tianjin's master thesis, 2007.6); Or the like;
2, reproducible wide host range plasmid, comprise: 1) pBBR1MCS (5144bp), its collection of illustrative plates is seen accompanying drawing 1 (literature reference Kovach, ME., Phillips, RW., Elzer, PH., et al.pBBR1MCS:a broad-host-range cloning vector, BioTechniques, 1994,16 (5): 800-802; Kovach ME, Elzer PH, Hill DS, et al.Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.Gene.1995; 166:175-176. can try to achieve); 2) pBBR1MCS-Pgap-FLP (6880bp), its structure is seen Fig. 2; Related primer P1 sees Table 1 to P4.
Table 1, structure primer
Genomic dna with zymomonas mobilis order-checking bacterial strain ZM4 is a template, according to the ZM4 whole genome sequence AE008692 design primer P1 and the P2 that have announced, amplification obtains 347bp, contains the product of the glyceraldehyde 3-phosphate dehydro-genase gene gap promoter region of ZM4, called after PCR1;
With the pCP20 plasmid is template, with the product that P3 and P4 increase and obtain 1474bp, contain yeast saccharomyces cerevisiae site-specific recombinase gene FLP, called after PCR2; (pCP20 sees document (Kirill A.Datsenko, Barry L.Wanner.One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products, Proc Natl Acad Sci U S A.2000June 6; 97 (12): 6640-6645))
Being template with PCR1 and PCR2 again, is primer with P1 and P4, and amplification obtains the product of 1801bp, and called after PCR3, PCR3 contain ClaI site, XbaI site and Pgap-FLP.
Press the specification sheets operation of the TA cloning vector pGEM-T easy test kit of Promega company, PCR3 is connected to pGEM-T easy, obtain plasmid pGEM-T easy-PCR3, sequencing result shows that sudden change does not take place, and the nucleotide sequence of Pgap-FLP is seen SEQ ID NO:1 in the sequence table.
PGEM-T easy-PCR3 is with the two fragments that obtain the 1783bp size of cutting of ClaI and XbaI, cuts big fragment (5095bp) and is connected connection product CaCl with the ClaI of pBBR1MCS-2 and XbaI are two
2The chemical method method transforms the Top10 competent cell, and with the screening plate screening of LB+Amp100 μ g/ml, the gained transformant extracts plasmid after with LB+Amp100 μ g/ml nutrient solution, carries out enzyme and cuts evaluation, proves to obtain pBBR1MCS-2-Pgap-FLP (6880bp).Be used to preserve and bacterial strain JM110 (pBBR1MCS-2-Pgap-FLP) the called after colon bacillus Escherichia coli of the pBBR1MCS-2-Pgap-FLP that increases, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date: on July 13rd, 2010, preserving number is CGMCC No.4003.
The plasmid type that can be used for the modification of zymomonas mobilis genomic dna has:
1, site-directed integration plasmid, as: pBR328-ldhR-cml-ldhL (7447bp), its collection of illustrative plates is seen Fig. 1, it makes up referring to document (Zou Shaolan, flood liberation, Ma Yuanyuan etc. the structure of zymomonas mobilis site-directed integration plasmid, Nankai University's journal (natural science edition), 2009,42 (6): 42-47); 2, swivel base plasmid at random.
DNA to be transformed can also be the dna fragmentation that does not contain any carrier, as endonuclease bamhi, PCR fragment.
The invention will be further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited only to this.If not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Electroporation transforms pBBR1MCS-2 (5144bp) to zymomonas mobilis bacterial strain ZM4 under embodiment 1, the different cell concentration multiple
Extract in a small amount from intestinal bacteria JM110 bacterial strain alkaline process and to obtain plasmid pBBR1MCS-2, the sterilized water dissolving is adjusted concentration and is 150ng/ μ l and is and obtains DNA to be transformed.
At first streak inoculation bacterial strain ZM4 is to RM solid medium flat board (2%D-glucose, 1%yeast extract, 0.2%KH
2PO
4, pH 6.0, and agar 1.8%), 30 ℃ leave standstill cultivation 3 days, carry out the flat board activation; The single bacterium colony of the dull and stereotyped growth of picking, inoculation RM liquid nutrient medium (2%D-glucose, 1%yeast extract, 0.2%KH
2PO
4, pH 6.0), 30 ℃ leave standstill cultivation 18 hours, are a liquid culture; The aforesaid liquid culture, by 1% inoculum size inoculation RM liquid, 30 ℃ leave standstill and are cultured to OD
600=0.405, ice bath 5min, get 3ml, 6ml, 12ml, 18ml, 24ml respectively in the 50ml centrifuge tube, 6000rpm, 4 ℃, the centrifugal collection thalline of 3min remove supernatant, the aseptic 10% glycerine washing of 10ml ice bath precooling, repeat 3 times, remove supernatant, each is resuspended with aseptic 10% glycerine of 120 μ l ice bath precoolings, and transfer in the aseptic centrifuge tube of 1.5ml, promptly obtain the competent cell that cycles of concentration is respectively 25 times, 50 times, 100 times, 150 times, 200 times.
Centrifuge tube at each competent cell adds above-mentioned DNA to be transformed 3 μ l, mixing, go in the 2mm electric shock cup of ice bath precooling, ice bath 5 minutes shocks by electricity under strength of electric field 11.75kV/cm on the electric conversion instrument (corresponding voltage is 2.35kV), electric capacity 25 μ F, resistance 200 Ω; Hit completely, add 30 ℃ 0.8ml RM (1%yeast extract, 2%D-glucose, 0.2%KH rapidly
2PO
4), 30 ℃ leave standstill 16 hours (recovery time); Bacterium liquid after the recovery is suitably centrifugal to be concentrated, coating is added with the RM screening dull and stereotyped (kalamycin final concentration 310 μ g/ml) of kalamycin, 30 ℃ leave standstill cultivation 5 days, the dull and stereotyped single bacterium colony of going up growth of picking screening, carrying out evaluation of RM+Km310 μ g/ml liquid culture and bacterium colony PCR identifies, and extract plasmid and carry out enzyme and cut, detect positive transformant, the statistics transformation efficiency.
Gained cycles of concentration and transformation efficiency corresponding relation the results are shown in Figure 3.For the highest, reach 1778/μ g DNA with the transformation efficiency of 100 times of cycles of concentration.
Electroporation transforms pZB21 (5930bp) to zymomonas mobilis bacterial strain CP4 under embodiment 2, the different cell concentration multiple
The method of pressing among the embodiment 1 is extracted plasmid pZB21, adjusts concentration and is 750ng/ μ l and be and obtain DNA to be transformed.
Press method activation, cultivation CP4 and preparation competent cell among the embodiment 1, not existing together is OD
600=0.40.Each adds above-mentioned DNA2 μ l to be transformed, with embodiment 1 operation, and strength of electric field 11.75kV/cm (corresponding voltage is 2.35kV), but recovery time 12h, microbiotic and concentration thereof are tsiklomitsin Tc1 7.5 μ g/ml.Gained cycles of concentration and transformation efficiency corresponding relation the results are shown in Figure 4.For the highest, reach 160/μ g DNA with the transformation efficiency of 100 times of cycles of concentration.
In embodiment 3, the electric shock cup under the different feeling attitude cell volume electroporation transform pBBR1MCS-2 (5144bp) to zymomonas mobilis bacterial strain ZM4
Plasmid is with embodiment 1, and the activation of ZM4, cultivation are with embodiment 1.OD
600=0.405 bacterium liquid, ice bath 5min, 6000rpm, 4 ℃, the centrifugal collection thalline of 3min, remove supernatant, the aseptic 10% glycerine washing of 20ml ice bath precooling, repeat 3 times, remove supernatant, with aseptic 10% glycerine of ice bath precooling resuspended (being that cycles of concentration is 100 times) of 1/100 volume of aforementioned nutrient solution volume; Carry out packing by 40 μ l, 80 μ l, 120 μ l, 160 μ l, 200 μ l in the aseptic centrifuge tube of each 1.5ml.
Ratio in the above-mentioned DNA to be transformed of the corresponding 1 μ l of per 40 μ l cells adds above-mentioned DNA to be transformed respectively in tubulature in above-mentioned minute, mixing, go in the 2mm electric shock cup of ice bath precooling, ice bath 10 minutes shocks by electricity under the strength of electric field 11.75kV/cm on the electric conversion instrument (corresponding voltage is 2.35kV); Hit completely, add 30 ℃ 0.8ml RM rapidly, 30 ℃ left standstill 20 hours, with embodiment 1 Screening and Identification.
Gained the results are shown in Figure 5, and is best with transformation efficiency under the 120 μ l cell volumes, reaches 2255/μ g DNA; Under 80 μ l and the 160 μ l also than higher.
Electroporation conversion pBBR1MCS-2 (5144bp), pZB21 (5930bp) and pBBR1MCS-2-Pgap-FLP (6880bp) are to ZM4 under embodiment 4, the different strength of electric field
The preparation of DNA to be transformed: pBBR1MCS-2 is with embodiment 1, and pZB21 is with embodiment 2, and pBBR1MCS-2-Pgap-FLP presses the preparation of embodiment 1 method, and adjusting concentration is 750ng/ μ l, is and obtains DNA to be transformed.Competent cell is with the method preparation of embodiment 3, and difference is: OD
600=0.42,100 times of spissated cells are by 120 μ l packing.The plasmid dosage is respectively: 3 μ l, 3 μ l, 2 μ l.11.0,11.75,12.5,13.25,14kV/cm (corresponding voltage be respectively 2.20,2.35,2.50,2.65,2.80kV) electroporation strength of electric field is respectively:.The recovery time is 23h.Kalamycin and tsiklomitsin working concentration are the same.Gained the results are shown in Figure 6, and all best with the 11.75kV/cm transformation efficiency, the highest transformation efficiency of three plasmids is respectively: 2424,18,194/μ g DNA; But more transformant is arranged all also under other strength of electric field.
Electroporation conversion pBBR1MCS-2 (5144bp), pZB21 (5930bp) and pZB21-Plac-lacZ (6375bp) are to CP4 under embodiment 5, the different strength of electric field
DNA to be transformed is that the preparation and the CP4 competent cell of plasmid prepares with embodiment 4 bacterium liquid OD
600=0.40, three plasmid concentrations are respectively: 150ng/ μ l, 750ng/ μ l and 750ng/ μ l.The plasmid dosage is respectively: 3 μ l, 2 μ l, 2 μ l.11.0,11.75,12.5,13.25kV/cm (corresponding voltage be respectively 2.20,2.35,2.50,2.65kV) electroporation strength of electric field is respectively:.The recovery time is 20h, 11h, 15h.Kalamycin and tsiklomitsin working concentration are the same.Gained the results are shown in Figure 7, and is all best with the 11.75kV/cm transformation efficiency, but more transformant is also all arranged under other strength of electric field.
Electroporation transforms pBR328-ldhR-cml-ldhL (7447bp) to ZM4 under embodiment 6, the different strength of electric field
Embodiment 1 legal system prepare plasmid pBR328-ldhR-cml-ldhL gets a part BanII linearization for enzyme restriction, ethanol sedimentation then; Enzyme is not cut sample and enzyme and is cut sample all to adjust concentration be 1 μ g/ μ l, is and obtains DNA to be transformed.Be equipped with competent cell and electroporation conversion by embodiment 4 legal systems, but bacterium liquid OD
600=0.402, the equal 3 μ l of DNA dosage to be transformed, strength of electric field sees Table 2, recovery time 15h.Use microbiotic paraxin Cm, its final concentration is 100 μ g/ml.The results are shown in Table 2.Table 2 shows: integration efficiency is the highest under the annular form plasmid 12.50kV/cm, and integration efficiency is the highest under the linear forms plasmid 13.25kV/cm, and linear forms are higher than annular form.
PBR328-ldhR-cml-ldhL integration efficiency under table 2, the different strength of electric field
Embodiment 7, electroporation transforms pBBR1MCS (5144bp), pZB21 (5930bp) to ZM4 under the different recovery time
With embodiment 4 legal system prepare plasmids and ZM4 competent cell, pBBR1MCS concentration 150ng/ μ l, pZB21 concentration 750ng/ μ l are and obtain DNA to be transformed, bacterium liquid OD=0.375, plasmid dosage 3 μ l, 2 μ l.Strength of electric field is 11.75kV/cm, and the recovery time is then selected 3h, 11h, 16h, 20h, 24h, 28h respectively.Gained the results are shown in Figure 8, and Fig. 8 shows: all there is higher transformation efficiency all recovery time of pBBR1MCS, but the highest relatively under the 24h, reaches 2604/μ gDNA; PZB21 also has transformant under all recovery time, but the highest with 11h, reaches 60/μ g DNA.
Plasmid preparation and the preparation of CP4 competent cell are with embodiment 4, and concentration is respectively: pBBR1MCS-2150ng/ μ l, pZB21750ng/ μ l and pZB21-Plac-lacZ 750ng/ μ l are and obtain DNA to be transformed.The plasmid dosage is respectively: 3 μ l, 1 μ l, 2 μ l.Electroporation strength of electric field is 11.75kV/cm (corresponding voltage is 2.35kV).The different recovery time is set.Kalamycin and tsiklomitsin working concentration are the same.Gained the results are shown in Figure 9, as seen under all recovery times transformant is arranged all, and pBBR1MCS-2 transformation efficiency under 20h is the highest, reaches 3422/μ g DNA; PZB21 transformation efficiency under 11h is the highest, reaches 168/μ g DNA; PZB21-Plac-lacZ transformation efficiency under 11h is the highest, reaches 125/μ g DNA.
Embodiment 9, ZM4 cell-free extract are handled pBBR1MCS-2 (5144bp) and pZB21-Plac-lacZ (6375bp), and electroporation is transformed into ZM4
The activation of ZM4 and cultivation are left standstill for 1,30 ℃ with embodiment and are cultured to OD
600=0.6.Get above-mentioned an amount of bacterium liquid, 8000rpm, 4 ℃, the centrifugal collection thalline of 3min, with the extract damping fluid HND washing of isopyknic ice bath precooling, the extract damping fluid HND of the ice bath precooling of 1/20 volume of usefulness nutrient solution volume is resuspended; The composition of extract damping fluid HND: the HEPES of pH7.0,200mM, 20mM Na
2EDTA, 5mM DTT; Ultrasonication broken wall on ice then, 14000rpm, 4 ℃, 8min are centrifugal, collect supernatant liquor, it is the zymomonas mobilis cell-free extract, and use at once, if also need preserve, then add the bovine serum albumin BSA (final concentration 0.1mg/ml) of sterile glycerol (final concentration 50%) and nuclease free; Select 100 μ l reaction systems, comprising: 100mM HEPES, pH=7.0,10mM Na
2EDTA, 2.5mM DTT, 5mM MgCl
2, 1 μ M SAM, 2～10 μ g DNA, 50 μ l zymomonas mobilis bacterial strain to be transformed cell-free extract reacted 3 hours down at 37 ℃.Reaction finishes, and carries out phenol: chloroform: Virahol (25: 24: 1, v: v: v) extracting and dehydrated alcohol deposition and purification, adjusted concentration and be respectively 150ng/ μ l, 750ng/ μ l by the sterilized water dissolving.
The competent cell that is equipped with the ZM4 zymomonas mobilis with embodiment 4 legal systems, bacterium liquid OD=0.41, strength of electric field is 11.75kV/cm, be 21h without the pBBR1MCS-2 of hatching with through recovery time of the pBBR1MCS-2 of hatching, be 12h without the pZB21-Plac-lacZ of hatching with through recovery time of the pZB21-Plac-lacZ of hatching.The gained transformation efficiency is followed successively by: 2294,6102,4,32/μ g DNA.Prove that it can obviously improve transformation efficiency.
The activation of ZM4 (ldh::FRT-cml-FRT) and cultivation are left standstill for 1,30 ℃ with embodiment and are cultured to OD
600=1.0.Get above-mentioned an amount of bacterium liquid, 12000,4 ℃, the centrifugal collection thalline of 6min, with the extract damping fluid HND washing of isopyknic ice bath precooling, the extract damping fluid HND of the ice bath precooling of 1/10 volume of usefulness nutrient solution volume is resuspended; The composition of extract damping fluid HND: the HEPES of pH7.0,200mM, 20mM Na
2EDTA, 5mM DTT; Ultrasonication broken wall on ice then, 18000rpm, 4 ℃, 12min are centrifugal, collect supernatant liquor, i.e. zymomonas mobilis cell-free extract; Select 100 μ l reaction systems, comprising: 100mM HEPES, pH=7.0,10mM Na2EDTA, 2.5mM DTT, 5mM MgCl
2, 1 μ M SAM, 2～10 μ g DNA, 50 μ l zymomonas mobilis bacterial strain to be transformed cell-free extract reacted 2 hours down at 37 ℃.Reaction finishes, and carries out phenol: chloroform: Virahol (25: 24: 1, v: v: v) extracting and dehydrated alcohol deposition and purification, adjusted concentration and be respectively 750ng/ μ l by the sterilized water dissolving.
Be equipped with ZM4 (ldh::FRT-cml-FRT) competent cell with embodiment 4 legal systems, but bacterium liquid OD=0.42, be 2 μ l without the pBBR1MCS-2-Pgap-FLP of hatching with through the plasmid dosage of the pBBR1MCS-2-Pgap-FLP of hatching.Strength of electric field is 11.75kV/cm, and the recovery time is then selected 3h, 11h, 16h, 21h respectively.Gained the results are shown in Table 3.
The pBBR1MCS-2-Pgap-FLP electroporation that table 3, ZM4 cell-free extract are handled is to ZM4 (individual/μ g DNA)
| The recovery time | ?3h? | 11h? | 16h? | 21h? |
| Without hatching | ?96? | 112? | 198? | 202? |
| Through hatching | ?528? | 517? | 1056? | 1030? |
Technical scheme of the present invention, be applicable to zymomonas mobilis ZM4 (=ATCC31821), CP4, ATCC10988 bacterial strain or ZM4 (ldh::FRT-cml-FRT).Bacterial strain ZM4 (=ATCC31821) can buy from U.S. ATCC with strains A TCC10988, CP4 can buy from Chinese Research for Industrial Microbial Germ preservation center C ICC, and it is numbered CICC10232.ZM4 (ldh::FRT-cml-FRT) is the derivative strain of ZM4, its building mode is seen document (Zou Shaolan, flood liberation, Ma Yuanyuan etc. the structure of zymomonas mobilis site-directed integration plasmid, Nankai University's journal (natural science edition), 2009,42 (6): 42-47) (pressing the described method of document, is that starting strain is bound to obtain ZM4 (ldh::FRT-cml-FRT) bacterial strain with ZM4).The present invention also is applicable to the ATCC10988 bacterial strain.
Claims (6)
1. the electroporation genetic manipulation method of a zymomonas mobilis, it is characterized in that comprising the steps: DNA to be transformed is transformed into by electroporation in the competent cell of zymomonas mobilis or with zymomonas mobilis cell-free extract and DNA to be transformed and hatch jointly, the DNA after will hatching by electroporation is transformed in the competent cell of zymomonas mobilis; The competent cell of described zymomonas mobilis is that zymomonas mobilis is cultured to OD
600nm=0.375～0.420 bacterium liquid concentrates 100 times and make; The parameter of electroporation operation is: the competent cell volume is 80 μ l～160 μ l in the 2mm electric shock cup; Strength of electric field is 11.0～14.0kV/cm; The recovery time is 3h～24h.
2. the electroporation genetic manipulation method of a kind of zymomonas mobilis as claimed in claim 1 is characterized in that described zymomonas mobilis cell-free extract is to make with following method: cultivate zymomonas mobilis to OD
600=0.6～1.0 nutrient solution, at 8000～12000rpm, 4 ℃, the centrifugal collection thalline of 3～6min, extract damping fluid HND washing with isopyknic ice bath precooling, extract damping fluid HND with the ice bath precooling of 1/20 to 1/10 volume of nutrient solution volume is resuspended, ultrasonic on ice broken wall, 14000～18000rpm, 4 ℃, 8～12min are centrifugal, collect supernatant liquor, described extract damping fluid HND is: the 200mM HEPES of pH=7.0,20mM Na
2EDTA, 5mM DTT.
3. the electroporation genetic manipulation method of a kind of zymomonas mobilis as claimed in claim 1, it is characterized in that described with zymomonas mobilis cell-free extract and DNA to be transformed hatch jointly for: select 100 μ l reaction systems, comprise 100mMHEPES, pH=7.0,10mM Na
2EDTA, 2.5mM DTT, 5mM MgCl
2, 1 μ M SAM, 2～10 μ g DNA to be transformed, 50 μ l zymomonas mobilis cell-free extracts, 37 ℃, reaction 2～3 hours, purifying, sterilized water dissolving.
4. as the electroporation genetic manipulation method of claim 1, a kind of zymomonas mobilis of 2 or 3, it is characterized in that described zymomonas mobilis is ZM4, CP4, ATCC10988 or ZM4 (ldh::FRT-cml-FRT).
5. the electroporation genetic manipulation method of a kind of motion Zymomonas mobilis as claimed in claim 1 is characterized in that the competent cell volume is 120 μ l in the described 2mm electric shock cup.
6. the electroporation genetic manipulation method of a kind of motion Zymomonas mobilis as claimed in claim 1 is characterized in that described strength of electric field is 11.75～13.25kV/cm.
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Non-Patent Citations (4)
| Title |
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| 《Bioprocess Engineering》 19980228 Ching-Chou Characteristics and transformation of Zymomonas mobilis with plasmid pKT230 by electroporation 第81-85页 1-6 第19卷, 第2期 * |
| 《中国优秀硕士学位论文全文数据库》 20050715 高卫华 运动发酵单胞菌和大肠杆菌间穿梭质粒载体的构建 第B018-11页 1-6 , 第3期 * |
| 《中国生物工程杂志》 20060831 赖伟坚 运动发酵单胞菌积累聚羟基丁酸提高乙醇产量 第52-56页 1-6 第26卷, 第8期 * |
| 《南开大学学报（自然科学版）》 20091231 邹少兰 等 运动发酵单胞菌定点整合质粒的构建 第42-47页 1-6 第42卷, 第6期 * |
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