CN116083466B - Construction method of genetically engineered blue algae with multiple sequences as homologous recombination sites - Google Patents
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Abstract
The invention belongs to the field of genetic engineering, and discloses a construction method of genetically engineered cyanobacteria by taking multiple complex sequences as homologous recombination sites, and particularly discloses a universal tool plasmid for expressing exogenous genes in synechocystis PCC6803 by taking the multiple complex sequences as homologous recombination sites, wherein the universal tool plasmid is provided with an upstream homologous arm and a downstream homologous arm which can carry out homologous recombination with the cyanobacteria genome multiple complex sequences as homologous recombination sites, and can recombine target genes into cyanobacteria genome; meanwhile, discloses a genetic engineering alga for producing cellulose endoenzyme CelI15, cellulose exoenzyme CBH II and xylanase xynLc by taking single repeated sequences in multiple repeated sequences or fragments thereof as upstream and downstream homology arms respectively through homologous recombination and a construction method.
Description
Technical Field
The invention belongs to the field of genetic engineering, discloses a construction method of genetically engineered cyanobacteria by taking multiple complex sequences as homologous recombination sites, and particularly discloses a universal tool plasmid for expressing exogenous genes in synechocystis PCC6803 by taking multiple complex sequences as homologous recombination sites.
Background
Blue algae is a kind of prokaryote capable of carrying out plant-type oxygen-producing photosynthesis, and Synechocystis PCC6803 is a single-cell blue algae with a natural transformation system, has the characteristics of high growth speed, simple culture condition, simple cell structure, clear genetic background, convenience in molecular operation and the like, can carry out autotrophic growth by utilizing light energy, can also carry out heterotrophic growth by utilizing glucose, and is one of important modes of research on photosynthetic molecular biology. In view of the advantages of easy purification of the exogenous gene product expressed by the blue algae, low cost of algae cell culture, difficult pollution and the like, the blue algae is used as an exogenous gene expression vector to produce high added value products such as medicines, biofuel and the like.
The blue algae is modified in molecular biology, a common plasmid is pBluescript SK and modified plasmids thereof, such as pBluescript SK T1T2 plasmid, which takes a light sensation promoter of a 510bp segment before a psbA2 gene initiation codon ATG of synechocystis PCC6803 as an homologous recombination vector promoter and also serves as an upstream arm, and takes a psbA2 gene ORF segment as a downstream arm, so that exogenous genes can be introduced into synechocystis PCC6803 and integrated into a genome. The target gene is introduced into the plasmid behind the 510bp fragment (light sensitive promoter) before the start codon ATG of the psbA2 gene, and the expression of the target gene is started by taking the 510bp light sensitive promoter fragment before the ATG of the psbA2 gene as the promoter of the target gene. However, the promoter is dependent on illumination, the promoter capability is weakened under the condition of no light, and the expression of the prototheca psbA2 gene is destroyed after the target gene is introduced, so that the promoter has a certain influence on the growth of the alga. More importantly, because only one recombination site exists in the algae-starting cell, the recombination success rate is low, and the growth speed of algae is obviously slower than that of escherichia coli (E.coli) and bacillus subtilis (Bacillus subtilis), so that the probability of obtaining successful transformants is low.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a construction tool plasmid of genetically engineered cyanobacteria taking multiple repeated sequences as homologous recombination sites and a construction method of genetically engineered cyanobacteria, and simultaneously discloses genetically engineered cyanobacteria which respectively takes fragments in single repeated sequences in the multiple sequences as upstream and downstream homology arms to obtain cellulose endoenzyme (CelI 15) through homologous recombination, wherein exogenous target genes in the method are not only limited to cellulose endoenzyme genes (CelI 15) but also include cellulose exoenzyme genes (CBH II) and xylanase genes (xynLc). The method can be used for blue algae with multiple repeated sequences in the whole genome sequence known at present, and exogenous genes are recombined on the genome by taking the multiple repeated sequences as homologous recombination sites.
The invention adopts the following technical scheme:
A universal plasmid is provided with an upstream homology arm and a downstream homology arm which can carry out homologous recombination by taking a multiple sequence of a blue algae genome as a homologous recombination site, and can transfer a biomass converting enzyme gene into the genome of the blue algae.
Further, a universal plasmid as described above may be shuttle expressed in E.coli and Synechocystis PCC 6803.
Further, a universal plasmid as described above wherein a single repeated complete fragment or partial fragment of a multiplex sequence is used as the upstream or downstream homology arm to the homologous recombination site.
Further, the universal plasmid is modified based on the pBluescriptSK plasmid, comprises a universal part of the pBluescriptSK plasmid, and has a sequence shown as SEQ ID NO. 44 in a sequence table; the modified part comprises an upstream homology arm, a downstream homology arm, a target gene, a resistance gene, a promoter and a terminator; the universal plasmid can integrate the target gene into the blue algae genome.
Furthermore, the promoter of the universal plasmid is a T7 promoter, the sequence of the universal plasmid is shown as SEQ ID NO. 37 in the list, the resistance gene of the universal plasmid is a Kana resistance gene, and the sequence of the universal plasmid is shown as SEQ ID NO. 34 in the list; or chloramphenicol resistance gene with sequence shown as SEQ ID NO. 33; or a spectinomycin resistance gene, the sequence of which is shown as SEQ ID NO:35 in the list.
Further, the above-mentioned universal plasmid, the multiple sequence refers to a base sequence which is present in the genome of the microorganism of interest to be modified by gene recombination and is repeated consecutively a plurality of times; wherein the continuous repetition number n is one selected from 2, 3, 4, 5, 6 and n is a natural number greater than 6, and the length of the single repeated base sequence is more than or equal to 200bp;
preferably, the number n of consecutive repetitions is selected from one of 4, 5, 6, and 6.
Further, in the above-mentioned universal plasmid, the length of the single repeated base sequence is in the range of 250bp to 1000bp, and the homology between the repeated base sequences is 97% or more.
Further, the above-mentioned universal plasmid is prepared by the following steps:
(1) The upstream homology arm is obtained by PCR amplification technology by taking SEQ ID NO. 1 and SEQ ID NO. 2 in a sequence table as upstream and downstream primers and taking a wild synechocystis PCC6803 genome as a template, so as to obtain the sequence shown as SEQ ID NO. in the sequence table: 31;
(2) The downstream homology arm is obtained by PCR amplification technology by taking SEQ ID NO 3 and SEQ ID NO 4 in a sequence table as upstream and downstream primers and taking a wild synechocystis PCC6803 genome as a template, so as to obtain the sequence shown as SEQ ID NO in the sequence table: shown at 32;
(3) The chloramphenicol resistance sequence is obtained by PCR amplification by taking SEQ ID NO. 5 and SEQ ID NO. 6 in the sequence table as upstream and downstream primers and PDK3 plasmid as a template, and the sequence is shown as SEQ ID NO. 33 in the sequence table; the KanR resistance sequence is obtained by PCR amplification by taking SEQ ID NO. 7 and SEQ ID NO. 8 in a sequence table as upstream and downstream primers and the Pet28a plasmid as a template, and the sequence is shown as SEQ ID NO. 34 in the sequence table; the resistance sequence of spectinomycin is obtained by PCR amplification by taking SEQ ID NO 9 and SEQ ID NO 10 in a sequence table as upstream and downstream primers and pEXT21 plasmid as a template, and the sequence is shown as SEQ ID NO 35 in the sequence table;
(4) Taking SEQ ID NO. 11 and SEQ ID NO. 12 in the sequence table as upstream and downstream primers, taking the escherichia coli genome as a template, and carrying out PCR amplification to obtain a T1T2 terminator sequence, wherein the sequence is shown as SEQ ID NO. 36 in the sequence table;
(5) The sequence of the T7 promoter is obtained by PCR amplification by taking SEQ ID NO. 13 and SEQ ID NO. 14 in a sequence table as upstream and downstream primers and taking a plasmid Pet28a as a template, and the sequence is shown as SEQ ID NO. 37 in the sequence table;
(6) Carrying out fusion PCR by taking SEQ ID NO. 1 and SEQ ID NO. 14 in the sequence table as upstream and downstream primers, and connecting a promoter with an upstream homology arm;
(7) Carrying out fusion PCR by taking SEQ ID NO. 5 and SEQ ID NO. 4 in the sequence table as upstream and downstream primers, and connecting chloramphenicol with downstream homology arms; carrying out fusion PCR by taking SEQ ID NO. 7 and SEQ ID NO. 4 in the sequence table as upstream and downstream primers, and connecting kanamycin with downstream homology arms; carrying out fusion PCR by taking SEQ ID NO 9 and SEQ ID NO 4 in the sequence table as upstream and downstream primers, and connecting spectinomycin with a downstream homology arm;
(8) The pBluescriptSK plasmid is subjected to PstI and BamHI double enzyme digestion to obtain a linearization vector;
(9) And (3) connecting the connecting fragment of the upstream homology arm and the promoter obtained in the step (6), the terminator fragment obtained in the step (4), the connecting fragment of chloramphenicol and the downstream homology arm or kanamycin and the downstream homology arm and spectinomycin and the downstream homology arm obtained in the step (7) and the vector obtained in the step (8) by utilizing recombinase ClonExpress MultiS One step Cloning Kit (Vazyme C113-02) to obtain recombinant expression vector plasmid pBluescriptSK-T7-MRS.
Further, the above-mentioned universal plasmid, the biomass converting enzyme gene is selected from one of a cellulose endoenzyme gene CelI15, a cellulose exoenzyme gene cbhii, and a xylanase gene xynLc.
Furthermore, the general plasmid is used in the construction method of the genetically engineered blue-green algae with multiple sequences as homologous recombination sites, the biomass converting enzyme gene is inserted between the upstream and downstream homology arms of the general plasmid, and then the genetically engineered blue-green algae is obtained through resistance screening.
Further, a pair of sequences useful for homologous recombination comprises an upstream homology arm sequence and a downstream homology arm sequence, wherein the upstream homology arm sequence is as set forth in SEQ ID NO:31, the downstream homology arm sequence is shown as SEQ ID NO: shown at 32.
Further, the above pair of sequences can be used for homologous recombination, which occurs in the synechocystis PCC 6803.
Furthermore, the genetically engineered blue algae contains the universal plasmid or is subjected to transformation treatment of the plasmid.
Furthermore, the initial strain of the genetically engineered cyanobacteria is derived from wild synechocystis PCC6803.
Furthermore, the genetically engineered cyanobacteria can be applied to the preparation of cellulose endo-enzyme, cellulose exo-enzyme or xylanase.
The invention has the beneficial effects that:
The construction method of genetically engineered blue algae with multiple recombination sequences as homologous recombination sites, and specifically discloses a universal tool plasmid for expressing exogenous genes in synechocystis PCC6803 by using the multiple recombination sequences as homologous recombination sites, wherein the universal plasmid can be expressed in E.coli and synechocystis PCC6803 in a shuttle manner; the invention uses T7 strong promoter to start the expression of target gene, uses a sequence of a 888bp continuous repeated sequence (base number 2354010-2359337 in genome) in the synechocystis PCC6803 as shown in SEQ ID NO:43 in a list, wherein, one sequence is used as the upper and lower homologous arms of the homologous recombination plasmid (the front 444bp of the single 888bp repeated sequence is used as the upper homologous arm and the rear 444bp is used as the lower homologous arm), and recombines the exogenous gene into the synechocystis PCC6803 genome through the homologous recombination site. Because the continuous repeated sequences are closely connected, a plurality of homologous recombination sites can be provided, and the recombination insertion probability of a target gene is increased, so that the probability of recombining an exogenous gene into the synechocystis PCC6803 is increased; the expression of the exogenous target gene in the synechocystis is independent of light, and the heterologous target gene can be expressed with high efficiency; the invention successfully integrates the cellulose endoenzyme gene CelI15, the cellulose exoenzyme gene CBH II and the xylanase gene xynLc to a segment of six-time repeated sequence in the synechocystis PCC6803 by using the method, thereby realizing the high-activity expression of the cellulose endoenzyme gene CelI15, the cellulose exoenzyme gene CBH II and the xylanase gene xynLc. Of course, the exogenous target gene in the method is not limited to the 3 genes, and the method can be used for blue algae with multiple repeated sequences in the whole genome sequence known at present, and the multiple repeated sequences are used as homologous recombination sites to recombine the exogenous gene on the genome.
Drawings
FIG. 1 is a basic structure diagram of a general plasmid according to the present invention;
FIG. 2 is a PCR detection chart of engineering algae strain P5S-MRS-CelI15, wherein lane 1 is Marker, lanes 2-9 all show bands at 1000-2000bp, and the bands are consistent with the expected base length 1503bp of CelI 15;
FIG. 3 is a graph showing how easily engineering strain P5S-MRS-CelI15 is transformed with engineering strain P5st1t2npt-CelI 15;
Wherein a is a transformant which appears at P5st1t2npt-CelI15 after 10days, and b is a transformant which appears at P5S-MRS-CelI after 10 days;
FIG. 4 is a graph showing comparison of cellulase activities of engineering strain and wild strain according to the present invention;
FIG. 5 is a schematic diagram of the insertion of homologous recombination target genes into multiple repeat sequence sites in the present invention;
FIG. 6 shows the expression of the target gene (measurement of cellulase activity) when the recombinant plasmid P5S-MRS-CelI15 of the present invention uses the Trc strong promoter.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely in the examples, and it is apparent that the described examples are only some of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents or instruments used in the examples of the present invention were not manufacturer-identified and were conventional reagent products commercially available.
Example 1
Construction of homologous recombinant plasmids P5S-MRS-CelI15, P5S-MRS-CBHII, P5S-MRS-xynLc and P5st1t2npt-CelI 15:
1. Construction of homologous recombinant plasmids P5S-MRS-CelI15, P5S-MRS-CBHII and P5S-MRS-xynLc9
(1) The upstream homology arm is obtained by PCR amplification technology by taking SEQ ID NO. 1 and SEQ ID NO. 2 in a sequence table as upstream and downstream primers and taking a wild synechocystis PCC6803 genome as a template, so as to obtain the sequence shown as SEQ ID NO. in the sequence table: 31;
(2) The downstream homology arm is obtained by PCR amplification technology by taking SEQ ID NO 3 and SEQ ID NO 4 in a sequence table as upstream and downstream primers and taking a wild synechocystis PCC6803 genome as a template, so as to obtain the sequence shown as SEQ ID NO in the sequence table: shown at 32;
(3) The chloramphenicol resistance sequence is obtained by PCR amplification by taking SEQ ID NO. 5 and SEQ ID NO. 6 in the sequence table as upstream and downstream primers and PDK3 plasmid as a template, and the sequence is shown as SEQ ID NO. 33 in the sequence table; the KanR resistance sequence is obtained by PCR amplification by taking SEQ ID NO. 7 and SEQ ID NO. 8 in a sequence table as upstream and downstream primers and the Pet28a plasmid as a template, and the sequence is shown as SEQ ID NO. 34 in the sequence table; the resistance sequence of spectinomycin is obtained by PCR amplification by taking SEQ ID NO 9 and SEQ ID NO 10 in a sequence table as upstream and downstream primers and pEXT21 plasmid as a template, and the sequence is shown as SEQ ID NO 35 in the sequence table;
(4) Taking SEQ ID NO. 11 and SEQ ID NO. 12 in the sequence table as upstream and downstream primers, taking the escherichia coli genome as a template, and carrying out PCR amplification to obtain a T1T2 terminator sequence, wherein the sequence is shown as SEQ ID NO. 36 in the sequence table;
(5) The sequence of the T7 promoter sequence obtained by PCR amplification by taking SEQ ID NO. 13 and SEQ ID NO. 14 in a sequence table as an upstream primer and a downstream primer and taking a plasmid Pet28a as a template is shown as SEQ ID NO. 37 in the list, the expression of a target gene can be started by a recombinant plasmid which is not any promoter in the recombinant plasmid, for example, the expression of the target gene is not successfully started by the recombinant plasmid constructed by a Trc strong promoter, and the enzyme activity measurement result is shown as figure 6;
(6) Taking SEQ ID NO. 15 and SEQ ID NO. 16 in the sequence table as upstream and downstream primers, taking a synthesized cellulose endoenzyme gene CelI15 from bacillus subtilis as a template (Genbank No. AY 044252), and obtaining a cellulose endoenzyme CelI15 gene fragment by a PCR amplification technology, wherein the sequence is shown as SEQ ID NO. 38 in the sequence table; the xylanase xynLc fragment is obtained by a PCR amplification technology by taking SEQ ID NO 17 and SEQ ID NO 18 in a sequence table as upstream and downstream primers and taking a synthesized xylanase gene xynLc from bacillus subtilis as a template, wherein the sequence is shown as SEQ ID NO 39 in the sequence table; the cellulose exo-enzyme CBHII fragment is obtained by PCR amplification technology by taking SEQ ID NO 19 and SEQ ID NO 20 in a sequence table as an upstream primer and a downstream primer and taking a CBHII gene of the Trichoderma reesei cellulose exo-glucanase as a template, wherein the sequence is shown as SEQ ID NO 40 in the sequence table;
(7) Carrying out fusion PCR by taking SEQ ID NO. 1 and SEQ ID NO. 14 in the sequence table as upstream and downstream primers, and connecting a promoter with an upstream homology arm;
(8) Carrying out fusion PCR by taking SEQ ID NO. 5 and SEQ ID NO. 4 in the sequence table as upstream and downstream primers, and connecting chloramphenicol with downstream homology arms; carrying out fusion PCR by taking SEQ ID NO. 7 and SEQ ID NO. 4 in the sequence table as upstream and downstream primers, and connecting kanamycin with downstream homology arms; carrying out fusion PCR by taking SEQ ID NO 9 and SEQ ID NO 4 in the sequence table as upstream and downstream primers, and connecting spectinomycin with a downstream homology arm;
(9) The pBluescriptSK plasmid is subjected to PstI and BamHI double enzyme digestion to obtain a linearization vector;
(10) Connecting the connecting segment of the upstream homology arm obtained in the step (7) and the promoter, the terminator segment obtained in the step (4), the connecting segment of chloramphenicol obtained in the step (8) and the downstream homology arm or kanamycin and the downstream homology arm and spectinomycin and the downstream homology arm, the target gene CelI15 segment or CBHII segment obtained in the step (6), xynLc segment and the vector obtained in the step (9) by utilizing recombinase ClonExpress MultiS One step Cloning Kit (Vazyme C113-02) to obtain recombinant expression vector plasmids P5S-MRS-CelI15, P5S-MRS-CBHII and P5S-MRS-xynLc;
(11) Naturally transforming the recombinant expression vector plasmids P5S-MRS-CelI15, P5S-MRS-CBHII and P5S-MRS-xynLc to synechocystis PCC6803 obtained in the step (10), performing resistance screening to obtain transgenic synechocystis, and culturing to obtain the strain named P5S-MRS-CelI15, P5S-MRS-CBHII and P5S-MRS-xynLc.
The double cleavage system used in this experiment was as follows:
the PCR reaction system and the procedure used in this experiment were as follows:
The procedure is as follows: pre-denaturation at 95℃for 3min,
Denaturation at 95℃for 15s,
Annealing at 56 deg.c for 15s,
The extension is carried out at 72 ℃ for 1Kb/30s,
Final extension at 72℃for 10min
Homologous recombinant plasmids P5S-MRS-CelI15, P5S-MRS-CBHII and P5S-MRS-xynLc9 were obtained through the above examples, and the general portions of the plasmids are shown in FIG. 1.
2. Construction of homologous recombinant plasmid p5st1t2npt-CelI15
(1) The sequence of the PsbA2 promoter and upstream homology arm is amplified by a PCR amplification technology by taking SEQ ID NO. 21 and SEQ ID NO. 22 in a sequence table as upstream and downstream primers and taking a wild synechocystis PCC6803 genome as a template, so as to obtain a sequence shown as SEQ ID NO. in the sequence table: 41;
(2) Taking SEQ ID NO. 23 and SEQ ID NO. 24 in the sequence table as upstream and downstream primers, taking a synthesized cellulose endoenzyme gene CelI15 from bacillus subtilis as a template (Genbank No. AY 044252), and obtaining a cellulose endoenzyme CelI15 gene fragment by a PCR amplification technology, wherein the sequence is shown as SEQ ID NO. 38 in the sequence table;
(3) Amplifying a PsbA2 downstream homology arm by using SEQ ID NO. 25 and SEQ ID NO. 26 in the sequence table as an upstream primer and a downstream primer and using a wild synechocystis PCC6803 genome as a template through a PCR amplification technology to obtain a sequence shown as SEQ ID NO. in the sequence table: 42;
(4) Taking SEQ ID NO. 27 and SEQ ID NO. 28 in the sequence table as upstream and downstream primers, taking the escherichia coli genome as a template, and carrying out PCR amplification to obtain a T1T2 terminator sequence, wherein the sequence is shown as SEQ ID NO. 36 in the sequence table;
(5) The KanR resistance sequence is obtained by PCR amplification by taking SEQ ID NO. 29 and SEQ ID NO. 30 in a sequence table as upstream and downstream primers and the Pet28a plasmid as a template; obtaining the sequence shown as SEQ ID NO in the sequence table: shown at 34;
(6) Fusion PCR is carried out by taking SEQ ID NO. 21 and SEQ ID NO. 24 in the sequence table as upstream and downstream primers, so as to obtain a connecting segment of the PsbA2 promoter and upstream homology arm and the target gene CelI 15;
(7) Fusion PCR is carried out by taking SEQ ID NO. 29 and SEQ ID NO. 26 in the sequence table as upstream and downstream primers to obtain a connecting segment of a downstream homology arm of KanR, T1T2 and PsbA 2;
(8) The pBluescriptSK plasmid is subjected to PstI and BamHI double enzyme digestion to obtain a linearization vector;
(9) And (3) connecting the PsbA2 promoter obtained in the step (6) and the connecting fragment of the upstream homology arm and the target gene CelI15, the KanR obtained in the step (7), the T1T2 and the connecting fragment of the PsbA2 downstream homology arm, the target gene CelI15 fragment obtained in the step (2) and the linearization vector obtained in the step (8) by utilizing recombinase ClonExpress MultiS One step Cloning Kit (Vazyme C113-02) to obtain homologous recombinant plasmid p5st1T2npt-CelI15.
(10) Naturally converting the recombinant expression vector plasmid p5st1t2npt-CelI15 obtained in the step (9) into synechocystis PCC6803, performing resistance screening to obtain transgenic synechocystis, and culturing to obtain the engineering strain p5st1t2npt-CelI 15.
Example 2
Acquisition of a genetically engineered strain of synechocystis PCC6803 capable of producing cellulases:
(1) Plasmid transformation
Taking 30ml of good algae liquid, centrifuging at 4deg.C and 5000r/min for 10min, discarding supernatant, adding fresh BG-11 culture medium, washing twice, re-suspending in 50ml of culture medium, and culturing to logarithmic phase (OD is 0.6-0.8). Taking 30ml of algae liquid in logarithmic growth phase, centrifuging at 4 ℃ for 10min at 5000r/min, adding fresh BG-11 culture medium to wash twice, washing once with 0.1mol/l CaCl2 and 0.01mol/NaCl solution, and adjusting OD to 2.5-3.5. Adding 500ul of algae solution into 40 mu L of recombinant plasmid, mixing, incubating (570 LUX) for 6 hours under weak light, shaking for 6 hours halfway, uniformly coating the algae solution on a non-anti-BG-11 solid flat plate with mixed fiber ester film placed thereon for 24 hours, preventing the mixed fiber ester film from being placed on the BG-11 solid flat plate with chloramphenicol concentration of 50ug/ml, and culturing for 7-10 days under 2000LUX light at 30 ℃ to start to appear transformants. After about two weeks, the transformant grows to a good state, and single algae are picked up and placed in a 5mlBG-11 liquid culture medium for culturing for one week, and the culture is gradually expanded, so that the later optimization is facilitated.
(2) Algae strain screening
The algae solution obtained in (1) was subjected to transfer culture under conditions of 29℃and 150rpm,1400Lux continuous light irradiation.
During transfer, the concentration of antibiotics in BG11 medium was increased to 20. Mu.g/mL. After the log phase is reached, transfer is carried out, and the concentration of antibiotics in the transfer culture medium is 10 mug/mL each time. When the antibiotic concentration in the medium reached 50. Mu.g/mL, the algae liquid plates were streaked. After the single algae grow out on the flat plate, the single algae are picked up and fall into the BG11 culture medium containing the corresponding antibiotic concentration for culture. After the algae grows to the logarithmic phase, the genome of the algae is extracted. Taking the genome as a template, and taking the genome as a template of SEQ ID NO:5 and SEQ ID NO: PCR was performed with primers 6. The PCR reaction system and procedure in the present invention were the same as in example 1. The PCR products were subjected to agarose electrophoresis, and the agarose gel detection chart is shown in FIG. 2. The strains selected are genetically engineered algae strains P5S-MRS-CelI15 and P5st1t2npt-CelI15, and the transformation difficulty is shown in figure 3.
Example 3
Expansion culture of Synechocystis PCC6803 genetically engineered algae strain P5S-MRS-CelI15 and P5st1t2npt-CelI 15:
Wild type Synechocystis PCC6803 is inoculated in 50mLBG-11 liquid medium, engineering strain P5S-MRS-CelI15 and P5st1t2npt-CelI15 are inoculated in 50mLBG-11 resistant liquid medium, OD 730=0.2 is regulated, and the strain is subjected to shaking culture (150 rpm) under the condition of 1400Lux continuous illumination at 28 ℃ for 7day.
Example 4
Cellulase enzyme activity assay of Synechocystis PCC6803 genetically engineered algae strain P5S-MRS-CelI15 and P5st1t2npt-CelI 15:
(1) Preparation of crude enzyme solution
The method comprises the steps of inoculating wild type synechocystis PCC6803 into 50mLBG-11 liquid medium, inoculating engineering strain P5S-MRS-CelI15 and P5st1t2npt-CelI15 into 50mLBG-11 resistant liquid medium, adjusting OD730 = 0.2, carrying out shake culture (150 rpm) to logarithmic phase under the condition of 1400Lux continuous illumination at 30 ℃, taking 30mL of algae liquid in logarithmic phase, centrifuging at 6000rpm for 10min, removing supernatant, re-suspending algae mud with fresh BG11 medium, centrifuging at 6000rpm for 10min, removing supernatant, collecting algae cells, and carrying out liquid nitrogen grinding and ultrasonic crushing on the collected algae cells to prepare crude enzyme liquid.
(2) DNS method measurement activity of cellulase
Taking 1.5mlCMC-Na solution and 0.5ml enzyme solution in a 25ml test tube, after the temperature is kept for 30min in a water bath at 40 ℃, immediately adding 1.5mlDNS color reagent, boiling in a 2ml1M/LNaOH boiling water bath for 5min, immediately cooling, adding distilled water, fixing the volume to 25ml, and measuring absorbance As under the condition of the wavelength of 540 nm.
Blank: firstly adding 1.5ml of DNS reagent, then adding 0.5ml of enzyme solution to be detected and 1.5ml of sodium carboxymethyl cellulose solution, boiling for 5min at 100 ℃ in a 25ml test tube, immediately cooling, adding redistilled water until the volume is 25ml, and measuring absorbance A at 540 nm.
Comparison of enzyme activities is shown in FIG. 4, and FIG. 5 shows the principle of homologous recombination in the above examples.
It should be understood that the above description is not intended to limit the invention to the particular embodiments disclosed, but to limit the invention to the particular embodiments disclosed, and that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications, adaptations, additions and alternatives falling within the spirit and scope of the invention.
Claims (3)
1. A universal plasmid is characterized in that the universal plasmid is provided with an upstream homologous arm and a downstream homologous arm which can carry out homologous recombination by taking a multiple sequence of a blue algae genome as a homologous recombination site, and can transfer a target gene into the blue algae genome;
the universal plasmid can be expressed in the escherichia coli and synechocystis PCC 6803 in a shuttle manner;
The general plasmid is prepared by the following steps:
(1) The upstream homology arm is obtained by PCR amplification technology by taking SEQ ID NO. 1 and SEQ ID NO. 2 in a sequence table as upstream and downstream primers and taking a wild synechocystis PCC6803 genome as a template, so as to obtain the sequence shown as SEQ ID NO. in the sequence table: 31;
(2) The downstream homology arm is obtained by PCR amplification technology by taking SEQ ID NO 3 and SEQ ID NO 4 in a sequence table as upstream and downstream primers and taking a wild synechocystis PCC6803 genome as a template, so as to obtain the sequence shown as SEQ ID NO in the sequence table: shown at 32;
(3) The chloramphenicol resistance sequence is obtained by PCR amplification by taking SEQ ID NO. 5 and SEQ ID NO. 6 in the sequence table as upstream and downstream primers and PDK3 plasmid as a template, and the sequence is shown as SEQ ID NO. 33 in the sequence table; the KanR resistance sequence is obtained by PCR amplification by taking SEQ ID NO. 7 and SEQ ID NO. 8 in a sequence table as upstream and downstream primers and the Pet28a plasmid as a template, and the sequence is shown as SEQ ID NO. 34 in the sequence table; the resistance sequence of spectinomycin is obtained by PCR amplification by taking SEQ ID NO 9 and SEQ ID NO 10 in a sequence table as upstream and downstream primers and pEXT21 plasmid as a template, and the sequence is shown as SEQ ID NO 35 in the sequence table;
(4) Taking SEQ ID NO. 11 and SEQ ID NO. 12 in the sequence table as upstream and downstream primers, taking the escherichia coli genome as a template, and carrying out PCR amplification to obtain a T1T2 terminator sequence, wherein the sequence is shown as SEQ ID NO. 36 in the sequence table;
(5) The sequence of the T7 promoter is obtained by PCR amplification by taking SEQ ID NO. 13 and SEQ ID NO. 14 in a sequence table as upstream and downstream primers and taking a plasmid Pet28a as a template, and the sequence is shown as SEQ ID NO. 37 in the sequence table;
(6) Carrying out fusion PCR by taking SEQ ID NO. 1 and SEQ ID NO. 14 in the sequence table as upstream and downstream primers, and connecting a promoter with an upstream homology arm;
(7) Carrying out fusion PCR by taking SEQ ID NO. 5 and SEQ ID NO. 4 in the sequence table as upstream and downstream primers, and connecting a chloramphenicol gene with a downstream homology arm; carrying out fusion PCR by taking SEQ ID NO. 7 and SEQ ID NO. 4 in the sequence table as upstream and downstream primers, and connecting a kanamycin gene with a downstream homology arm; carrying out fusion PCR by taking SEQ ID NO 9 and SEQ ID NO 4 in the sequence table as upstream and downstream primers, and connecting a spectinomycin gene with a downstream homology arm;
(8) The pBluescriptSK plasmid is subjected to PstI and BamHI double enzyme digestion to obtain a linearization vector, the sequence of which is shown as SEQ ID NO. 44 in a sequence table;
(9) And (3) connecting the connecting fragment of the upstream homology arm obtained in the step (6) and the promoter, the terminator fragment obtained in the step (4), the connecting fragment of chloramphenicol obtained in the step (7) and the downstream homology arm or kanamycin and the downstream homology arm or spectinomycin and the downstream homology arm with the vector obtained in the step (8) by utilizing recombinase ClonExpress MultiS One step Cloning Kit to obtain recombinant expression vector plasmid pBluescriptSK-T7-MRS.
2. A construction method of genetically engineered blue algae using multiple sequences as homologous recombination sites is characterized in that the universal plasmid as claimed in claim 1 is used, a biomass converting enzyme gene is inserted between upstream and downstream homology arms of the universal plasmid, and then the genetically engineered blue algae is obtained by transferring the genetically engineered blue algae into blue algae cells and performing resistance screening.
3. The method for constructing genetically engineered cyanobacteria with multiple sequences as homologous recombination sites according to claim 2, wherein the biomass converting enzyme is one selected from the group consisting of an endo-cellulose enzyme CelI15, an exo-cellulose enzyme CBH ii and a xylanase xynLc.
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