WO2021249536A1 - 含barstar基因的工程菌及其在barnase基因克隆中的应用 - Google Patents

含barstar基因的工程菌及其在barnase基因克隆中的应用 Download PDF

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WO2021249536A1
WO2021249536A1 PCT/CN2021/099659 CN2021099659W WO2021249536A1 WO 2021249536 A1 WO2021249536 A1 WO 2021249536A1 CN 2021099659 W CN2021099659 W CN 2021099659W WO 2021249536 A1 WO2021249536 A1 WO 2021249536A1
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gene
sequence
barstar
plasmid
engineered
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PCT/CN2021/099659
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English (en)
French (fr)
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陈康明
周斌
毛灵琪
孙迎娣
孟庆伟
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南京金斯瑞生物科技有限公司
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Priority to CN202180041549.3A priority Critical patent/CN115698271A/zh
Publication of WO2021249536A1 publication Critical patent/WO2021249536A1/zh

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of genetic engineering, and specifically relates to an engineered bacteria containing a barstar gene and a construction method thereof, and the application of the engineered bacteria in the cloning of the ribonuclease barnase gene.
  • Barnase is a 12kD extracellular small molecule RNase produced by Bacillus amyloliquefaciens. It can degrade RNA in cells and is highly toxic.
  • the Bacillus amyloliquefaciens genome also contains the barnase specific antagonist gene barstar, and its product (10kD) can specifically bind to Barnase at a ratio of 1:1 to form a highly stable complex, thereby causing the Barnase expressed by the bacteria to lose the enzyme.
  • Activity [1] Taking advantage of the feature that its expression in specific cells will cause cell death, scientists use plant fertility or disease resistance-related promoters to regulate the activity of barnase in cells, thereby achieving male sterility and increased disease resistance of plants .
  • barnase also has many other important biological functions, such as controlling tumor angiogenesis, killing tumor cells and inhibiting the replication of viruses (including HIV virus) [3] .
  • viruses including HIV virus
  • barnase and barstar are cloned on the same plasmid vector [6] .
  • the disadvantage of this method is that there are additional barstar genes on the final plasmid, which affects subsequent downstream applications.
  • the third is to insert an intron sequence inside the barnase gene, so that the barnase expressed in E. coli contains additional sequences and loses its toxicity, and the intron sequence is removed from eukaryotic cells to restore its activity [7] .
  • the disadvantage of this method is that there is no intron sequence common to eukaryotic cells.
  • barnase cloning technology has a low success rate, cannot meet downstream applications, lacks universal intron sequences and other shortcomings, it is impossible to realize all the scenarios of barnase gene cloning.
  • conventional engineering bacteria are genetically modified through CRISPR-Cas9 technology.
  • the barstar gene expression element is inserted into the genome of the engineered bacteria, so that the Barstar expressed on the genome can neutralize the toxic effect of the barnase expressed on the plasmid, so that gene synthesis, plasmid construction and plasmid extraction can be successfully completed.
  • One aspect of the present invention provides an engineered bacterium, characterized in that the chromosomal genome of the engineered bacterium contains a barstar gene, and the engineered bacterium can be used to clone the barnase gene.
  • the barstar gene in the present invention comprises the nucleotide sequence shown in SEQ ID NO:1, or a sequence that is at least 80% identical to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the barstar gene comprises at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95% of the nucleotide sequence shown in SEQ ID NO:1. , A sequence with at least 97% or at least 99% identity. In other embodiments, the barstar gene comprises a sequence that is at least 91% identical to the nucleotide sequence shown in SEQ ID NO:1.
  • the barstar gene comprises a sequence that is at least 93% identical to the nucleotide sequence shown in SEQ ID NO:1. In other embodiments, the barstar gene comprises a sequence that is at least 95% identical to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the barstar gene comprises a sequence that is at least 97% identical to the nucleotide sequence shown in SEQ ID NO:1. In other embodiments, the barstar gene comprises a sequence that is at least 99% identical to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the barstar gene comprises the nucleotide sequence shown in SEQ ID NO:1. In a specific embodiment, the nucleotide sequence of the barstar gene is shown in SEQ ID NO:1.
  • the barstar gene is derived from Bacillus amyloliquefaciens.
  • the chromosomal genome of the engineered bacteria includes an expression element containing a barstar gene.
  • the barstar gene expression element includes a promoter and a terminator.
  • the barstar gene expression element comprises a continuously expressed promoter.
  • the barstar gene expression element comprises a promoter selected from one or more of EM7, recA, trp, araBAD, TEF1, GAL1, GAL10, lac, psbA, T7 or tacP promoter;
  • the terminator contained in the barstar gene expression element is selected from one or more of MrrnB T1, T7Te, rrnBT1, rrnBT2, rrnB1 or rrnB2 terminator.
  • the promoter is the EM7 promoter
  • the terminator is the MrrnB T1 terminator and the T7Te terminator.
  • the barstar gene expression element comprises the nucleotide sequence shown in SEQ ID NO: 2, or a sequence that is at least 80% identical to the nucleotide sequence shown in SEQ ID NO: 2. In other embodiments, the barstar gene expression element comprises at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, and the nucleotide sequence shown in SEQ ID NO: 2 A sequence that is at least 95%, at least 97%, or at least 99% identical. In some embodiments, the barstar gene expression element comprises a sequence that is at least 91% identical to the nucleotide sequence shown in SEQ ID NO: 2.
  • the barstar gene expression element comprises a sequence that is at least 93% identical to the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the barstar gene expression element comprises a sequence that is at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 2. In other embodiments, the barstar gene expression element comprises a sequence that is at least 97% identical to the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the barstar gene expression element comprises a sequence that is at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 2. In other embodiments, the barstar gene expression element comprises the nucleotide sequence shown in SEQ ID NO:2. In a specific embodiment, the nucleotide sequence of the barstar gene expression element is shown in SEQ ID NO: 2.
  • the engineered bacteria is selected from bacteria or fungi; preferably, the bacteria is selected from Escherichia coli or Bacillus subtilis, and the fungus is selected from yeast; more preferably, the E. coli is selected from TOP10F , JM108, DH5a, stbl3, JM109, DH10b, EPI300 or EPI400.
  • the engineered bacteria is Escherichia coli.
  • the engineered bacteria is Bacillus subtilis.
  • the E. coli engineered bacteria is selected from TOP10F, JM108, DH5a, stbl3, JM109, DH10b, EPI300 or EPI400.
  • the engineered bacteria is selected from TOP10F or JM108.
  • the engineered bacteria is TOP10F.
  • the engineered bacteria is JM108.
  • the barstar gene is cloned into the chromosome of E. coli engineered bacteria lacZ, recA, araD, dam, galE, galU, malA, ompT, tonA, rha or CyaA gene downstream; preferably cloned into E. coli engineering Downstream of the CyaA gene in the bacterial chromosome.
  • the barstar gene is cloned downstream of the CyaA gene in the E. coli engineered chromosome.
  • the barstar gene expression element is cloned into the chromosome of E. coli engineered bacteria lacZ, recA, araD, dam, galE, galU, malA, ompT, tonA, rha or CyaA gene downstream; preferably cloned into the large intestine Downstream of the CyaA gene in the chromosome of Bacillus engineering bacteria.
  • the barstar gene expression element is cloned downstream of the CyaA gene in the chromosome of the E. coli engineered bacteria.
  • Another aspect of the present invention provides a method for preparing engineered bacteria, which is characterized in that the barstar gene is cloned into the chromosomal genome of the engineered bacteria through gene editing technology.
  • the barstar gene comprises the nucleotide sequence shown in SEQ ID NO:1, or a sequence that is at least 80% identical to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the barstar gene comprises at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95% of the nucleotide sequence shown in SEQ ID NO:1. , A sequence with at least 97% or at least 99% identity. In other embodiments, the barstar gene comprises a sequence that is at least 91% identical to the nucleotide sequence shown in SEQ ID NO:1.
  • the barstar gene comprises a sequence that is at least 93% identical to the nucleotide sequence shown in SEQ ID NO:1. In other embodiments, the barstar gene comprises a sequence that is at least 95% identical to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the barstar gene comprises a sequence that is at least 97% identical to the nucleotide sequence shown in SEQ ID NO:1. In other embodiments, the barstar gene comprises a sequence that is at least 99% identical to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the barstar gene comprises the nucleotide sequence shown in SEQ ID NO:1. In a specific embodiment, the nucleotide sequence of the barstar gene is shown in SEQ ID NO:1.
  • the engineered bacterial chromosome genome further includes an expression element containing a barstar gene cloned by gene editing technology, and the barstar gene expression element includes a promoter and a terminator.
  • the barstar gene expression element comprises a promoter selected from one or more of EM7, recA, trp, araBAD, TEF1, GAL1, GAL10, lac, psbA, T7 or tacP promoter;
  • the terminator contained in the barstar gene expression element is selected from one or more of MrrnB T1, T7Te, rrnBT1, rrnBT2, rrnB1 or rrnB2 terminator.
  • the promoter is the EM7 promoter
  • the terminator is the MrrnB T1 terminator and the T7Te terminator.
  • the barstar gene expression element comprises the nucleotide sequence shown in SEQ ID NO: 2, or a sequence that is at least 80% identical to the nucleotide sequence shown in SEQ ID NO: 2. In other embodiments, the barstar gene expression element comprises at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, and the nucleotide sequence shown in SEQ ID NO: 2 A sequence that is at least 95%, at least 97%, or at least 99% identical. In some embodiments, the barstar gene expression element comprises a sequence that is at least 91% identical to the nucleotide sequence shown in SEQ ID NO: 2.
  • the barstar gene expression element comprises a sequence that is at least 93% identical to the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the barstar gene expression element comprises a sequence that is at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 2. In other embodiments, the barstar gene expression element comprises a sequence that is at least 97% identical to the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the barstar gene expression element comprises a sequence that is at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 2. In other embodiments, the barstar gene expression element comprises the nucleotide sequence shown in SEQ ID NO: 2. In a specific embodiment, the nucleotide sequence of the barstar gene expression element is shown in SEQ ID NO: 2.
  • the gene editing technology is selected from CRISPR-Cas9 technology, zinc finger nuclease technology or transcription activator-like effector nuclease technology.
  • the gene editing technology is CRISPR-Cas9 technology.
  • the gene editing technology is CRISPR-Cas9 technology, which includes the following steps:
  • step (2) The plasmid obtained in step (1) and the pCas9 plasmid are co-transfected into engineered bacterial cells, and incubated;
  • step (2) the cultivated engineered bacteria are screened for resistance to obtain a strain in which the barstar gene is integrated into the chromosome genome of the engineered bacteria.
  • the engineered bacteria is Escherichia coli or Bacillus subtilis; preferably, the engineered bacteria are selected from TOP10F, JM108, DH5a, stbl3, JM109, DH10b, EPI300 or EPI400; more preferably, The engineered bacteria is TOP10F or JM108. In some embodiments, the engineered bacteria is Escherichia coli. In other embodiments, the engineered bacteria is Bacillus subtilis. In some embodiments, the E. coli engineered bacteria is selected from TOP10F, JM108, DH5a, stbl3, JM109, DH10b, EPI300 or EPI400.
  • the engineered bacteria is selected from TOP10F or JM108. In one embodiment, the engineered bacteria is TOP10F. In another embodiment, the engineered bacteria is JM108.
  • the barstar gene is cloned into the downstream of the lacZ, recA, araD, dam, galE, galU, malA, ompT, tonA, rha or CyaA gene in the E. coli engineered chromosome. In some embodiments, the barstar gene is cloned downstream of the CyaA gene in the E. coli engineered chromosome.
  • the barstar gene expression element is cloned into the downstream of the lacZ, recA, araD, dam, galE, galU, malA, ompT, tonA, rha or CyaA gene in the E. coli engineered chromosome.
  • the barstar gene expression element is cloned downstream of the CyaA gene in the chromosome of the E. coli engineered bacteria.
  • coli engineered bacteria is selected from the downstream sequence of lacZ, recA, araD, dam, galE, galU, malA, ompT, tonA, rha or CyaA gene; preferably The target sequence cloned into the E. coli engineered bacteria gene is the downstream sequence of the CyaA gene.
  • the gRNA in the step (1) is designed according to the gene locus of the barstar gene cloned into the engineered bacteria.
  • the gRNA targeting sequence in the step (1) (the barstar gene is cloned into the engineered bacteria gene target sequence) is a 1-200bp sequence located downstream of the stop codon of the CyaA gene on the genome of the E. coli engineered bacteria.
  • the gRNA targeting sequence in the step (1) is a sequence of 1-100 bp downstream of the stop codon of the CyaA gene of the E. coli engineered bacteria genome.
  • the gRNA targeting sequence in the step (1) is a 1-50 bp sequence downstream of the stop codon of the CyaA gene on the genome of the E. coli engineered bacteria.
  • the gRNA targeting sequence in the step (1) is a 10-30 bp sequence downstream of the stop codon of the CyaA gene on the genome of the engineered Escherichia coli bacteria.
  • the sequence adjacent to the 3'end of the gRNA targeting sequence is NGG.
  • the sequence length of the gRNA in the step (1) is 10-30 bp, preferably, the sequence length of the gRNA is 20 bp.
  • the gRNA includes at least one sequence, preferably at least two sequences, and more preferably three sequences. In other embodiments, the gRNA comprises a sequence that is at least 80% identical to the nucleotide sequence shown in SEQ ID NO: 7, 8 or/and 9. In some embodiments, the gRNA comprises at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93% of the nucleotide sequence shown in SEQ ID NO: 7, 8 or/and 9. %, at least 95%, at least 97%, or at least 99% identical sequences. In a specific embodiment, the gRNA comprises the nucleotide sequence shown in SEQ ID NO: 7, 8 and 9.
  • the pTarget plasmid containing the gRNA sequence in the step (1) is obtained by mutating and amplifying the empty vector pTarget plasmid as a template.
  • the left and right homologous arm sequences in the step (1) are obtained by amplifying the 50-500 bp sequence on the left and right sides of the target sequence of the engineered bacteria.
  • the left and right homologous sequences synthesized in the step (1) are amplified and fused with the barstar gene expression element sequence, and then inserted into the pTarget plasmid containing the gRNA sequence.
  • the step (1) includes: a.
  • pTarget plasmid Use the empty vector pTarget plasmid as a template to perform mutation and amplify to obtain a pTarget plasmid containing gRNA; b. Select a sequence of 50-500 bp based on the left and right sides of the gRNA target sequence to perform PCR Amplify to obtain the left and right homologous arm sequences of homologous recombination; c. The homologous arm sequence and the barstar expression element sequence are fused, and then inserted into the pTarget-gRNA plasmid, after sequencing and verification, the sequence containing gRNA and barstar expression element is obtained PTarget plasmid.
  • the step (1) includes performing point mutation amplification using the empty vector pTarget plasmid as a template to obtain a pTarget plasmid containing gRNA, and then selecting a 500bp sequence on the left and right sides of the gRNA target sequence on the genome of the engineered bacteria.
  • PCR amplification the left and right homologous arm sequences of homologous recombination were obtained; the homologous arm sequence and the barstar expression element sequence were fused by PCR, and the obtained product was inserted into the pTarget-sgRNA plasmid through the EcoRI-HindIII restriction site and sequenced
  • a pTarget plasmid containing gRNA and barstar expression element sequences was obtained.
  • the plasmid obtained in step (1) and the pCas9 plasmid of step (2) each contain at least one resistance gene.
  • the gRNA-containing plasmid and the resistance gene of the pCas9 plasmid are selected from the group consisting of spectinomycin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene or ampicillin resistance, respectively Gene.
  • the gRNA-containing plasmid contains a spectinomycin resistance gene
  • the pCas9 plasmid contains a kanamycin resistance gene.
  • the step (2) includes firstly electrotransforming the pCas9 plasmid with the first resistance into the engineered bacteria at 30°C, and then selecting the first resistance-containing medium to obtain a successful transfer.
  • the strain with pCas9 plasmid is activated overnight in the liquid medium containing the first resistance, and then transferred to the liquid medium containing the first resistance according to the ratio of 1:100-1000 (V:V), and cultivated After a period of time, add 1ml arabinose of 1:1-100 (V:V) to induce cell growth until the OD600 is 0.4-0.6.
  • the first resistance and the second resistance are selected from the group consisting of spectinomycin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene or ampicillin resistance gene.
  • the step (2) includes first electrotransforming the Kan-resistant pCas9 plasmid into the engineered bacteria at 30°C, and then screening in the medium containing Kan to obtain the successfully transformed pCas9 plasmid Strains, activate the strains in liquid medium containing Kan overnight, and transfer them to liquid medium containing Kan at a ratio of 1:200-800 (V:V). After culturing for a period of time, add 1:100(V) :V) 1ml arabinose induce cell growth until the OD600 is 0.4-0.6.
  • step (1 ) The obtained pTarget plasmid containing Spec resistance was electrotransformed, and liquid medium was added to activate after electrotransformation; the strain was cultured overnight in the medium containing Kan and Spec, and finally PCR bacterial detection was performed to screen out that the barstar was introduced into the genome. Gene-positive strains.
  • the step (2) includes electrotransforming the Kan-resistant pCas9 plasmid into Escherichia coli at a temperature of 30°C, and then screening in a medium containing Kan to obtain the successful transfer of the pCas9 plasmid
  • the strain was activated overnight in a liquid medium containing Kan, and then transferred to a liquid medium containing Kan at a ratio of 1:400 (V:V). After culturing for a period of time, add 1:100 (V: V) 1ml arabinose induces cell growth until the OD600 is 0.4-0.6.
  • step (1) The obtained pTarget plasmid containing Spec resistance was electrotransformed, and liquid medium was added to activate after electrotransformation; the strain was cultured overnight in the medium containing Kan and Spec, and finally PCR was performed to screen out the barstar gene introduced into the genome. Positive strains.
  • the step (2) includes first electrotransforming the Kan-resistant pCas9 plasmid into TOP10F or JM108 at a temperature of 30°C, and then screening in a medium containing Kan to obtain successful transfer to pCas9 Plasmid strain, activate the strain in liquid medium containing Kan overnight, and then transfer it to liquid medium containing Kan at a ratio of 1:400 (V:V). After culturing for a period of time, add 1:100 (V) :V) 1ml arabinose induces cell growth until the OD 600 is 0.4-0.6.
  • step ( 1) The obtained pTarget plasmid containing Spec resistance was electrotransformed, and then liquid medium was added to activate after electrotransformation; the strain was cultured overnight in the medium containing Kan and Spec, and finally PCR bacterial detection was performed to screen out the introduction of the genome A positive strain of the barstar gene.
  • the resistance screening in the step (3) refers to the screening of engineered strains in which the barstar gene is integrated into the chromosomal genome through a double-resistant medium. In some embodiments, the resistance screening in the step (3) is to screen out engineered strains in which the barstar gene is integrated into the chromosomal genome through a medium containing one or both of Kan or Spec resistance.
  • the step (3) includes picking a single colony of the positive engineered bacteria obtained in step (2) and inoculating it into a medium containing the first resistance, adding 0.1-1mM IPTG to induce strain expression, and then The strains were respectively inoculated in two mediums containing the first resistance and those containing the second resistance. After culturing at 30°C, the strains were selected to be unable to grow in the medium containing the second resistance.
  • the normal-growing strain is the strain that has eliminated the pTarget plasmid.
  • the strain is then inoculated multiple times in non-resistant medium and incubated at 37°C, and then the strains are respectively inoculated to contain the first resistance and the second In the two resistant media, when the strains do not grow in the two media respectively, an engineered strain that eliminates pCas9 and pTarget is obtained.
  • the step (3) includes picking a single colony of the positive engineered bacteria obtained in step (2) and inoculating it into a medium containing Kan, adding 0.1-0.8mM IPTG to induce the expression of the strain, and then the strain Separately inoculate in two mediums containing Spec and Kan. After culturing at 30°C, select the strains that cannot grow on the medium containing Spec but grow normally on the medium containing Kan, that is, the pTarget plasmid is eliminated Inoculate the strains in non-resistant liquid medium several times. After culturing at 37°C, inoculate the strains into two mediums containing Kan and Spec. When the strains are in the two mediums When neither of them grows, an engineered strain that eliminates pCas9 and pTarget is obtained.
  • the step (3) includes picking a single colony of the Escherichia coli obtained in step (2) and inoculating it into Kan-containing LB medium, adding 0.5mM IPTG to induce the expression of the strain, and then dividing the bacterial solution separately Inoculated in two LB mediums containing Spec and Kan. After culturing at 30°C, the strains that cannot grow in the medium containing Spec but grow normally in the medium containing Kan are selected as the strains that have eliminated the pTarget plasmid. Then the strains were inoculated multiple times in non-resistant LB medium, and after culturing at 37°C, the strains were then inoculated into two LB mediums containing Kan and Spec. When the strains were both in the two mediums When not growing, an engineered strain that eliminates pCas9 and pTarget is obtained.
  • the step (3) preferably includes inoculating a single colony of TOP10F or JM108 obtained in step (2) into LB medium containing Kan, and adding 0.5 mM IPTG to induce strain expression, Then the strains were cultured in two LB mediums containing Spec and Kan, and cultured at 30°C, and the strains that could not grow in the Spec-containing medium but the normal growth containing Kan were selected to eliminate the pTarget plasmid. Then inoculate the strains in non-resistant LB liquid medium, cultivate them at 37°C, and then inoculate the strains into two LB mediums containing Kan and Spec. When the strains are in the two mediums Without growth respectively, an engineered strain that eliminates pCas9 and pTarget is obtained.
  • Another aspect of the present invention provides a method for cloning a barnase gene, which is characterized in that the method comprises transfecting a plasmid containing the barnase gene into the above-mentioned engineered bacteria for culture and amplification.
  • the present invention also provides a method for cloning the barnase gene, which is characterized in that the method comprises transforming the plasmid containing the barnase gene into the engineered bacteria prepared by the above method, culturing and amplifying it.
  • the above method for cloning the barnase gene further includes screening the engineered bacteria that transform the barnase gene through a double-resistant medium.
  • the engineered bacteria transformed with the barnase gene are obtained through Kan resistance and/or Spe resistance medium screening.
  • the engineered bacteria transformed with the barnase gene can be obtained through screening of Kan resistance and Spe resistance medium.
  • the method further includes performing plasmid extraction on the amplified and cultured engineered bacteria.
  • the method further includes sequence verification of the extracted plasmid.
  • the plasmid containing the barnase gene is selected from the group consisting of prokaryotic expression vector pET, pGEX series, yeast vector pRS, pESC series, plant vector pCAMBIA series, eukaryotic expression vector pcDNA3.1 series, pUC57, U9041-1 or U9041-12. In some preferred embodiments, the plasmid pUC57, U9041-1 or U9041-12 containing the barnase gene.
  • the "engineered bacteria” in the present invention can be any prokaryotic or eukaryotic microorganisms.
  • the prokaryotic microorganisms can be bacteria, including but not limited to Escherichia coli and Bacillus subtilis.
  • the eukaryotic microorganisms include but are not limited to yeast, Aspergillus.
  • the engineered bacteria is Escherichia coli or Bacillus subtilis.
  • E. coli can be selected from TOP10F, JM108, DH5a, stbl3, JM109, DH10b, EPI300 or EPI400.
  • the engineered bacteria is TOP10F or JM108.
  • Donor sequence refers to the artificial construction of a sequence in homologous recombination that contains a certain length (for example, 50-500bp) homologous sequence upstream and downstream of the targeted site, so that the target Insertion or deletion of a sequence to the site by means of homologous recombination.
  • a certain length for example, 50-500bp
  • homologous arm sequence refers to a DNA fragment with the same sequence as the upstream and downstream of the targeting sequence, which is used to identify the region where the targeting sequence undergoes homologous recombination, and is generally 50-500 bp in length.
  • vector is used herein to refer to a nucleic acid molecule capable of transferring or delivering another nucleic acid molecule.
  • the transferred nucleic acid is usually linked to the carrier nucleic acid molecule, for example, inserted into the carrier nucleic acid molecule.
  • the vector may include sequences that direct autonomous replication in the cell, or may include sufficient sequences to allow integration into the host cell's DNA.
  • Useful vectors include, for example, plasmids (such as DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.
  • transfection refers to the transfer of a polynucleotide or other biologically active compound from outside the cell to the inside of the cell so that the polynucleotide or biologically active compound is functional.
  • transfection reagents used to deliver polynucleotides to cells in vitro include, but are not limited to: liposomes, lipids, polyamines, calcium phosphate precipitation, histones, polyaziridines, and amphoteric polyelectrolyte complexes, and A combination of these.
  • Many in vitro transfection reagents are cationic, which allows the reagent to bind or form complexes with negatively charged nucleic acids through electrostatic interactions.
  • Co-transfection refers to the transfer of two or more plasmids into the same competent cell. Commonly used transfection methods in this field can be used, including electroporation, chemical transfection and other methods to transfer two or more plasmids into the same competent cell. The same competent cell. In the present invention, the electrotransformation method is preferred, in which two or more plasmids are transferred into the same competent cell.
  • Competent cell refers to the induction of cells through physical and chemical methods to absorb DNA molecules in the surrounding environment, so that they are in a physiological state that is optimal for ingesting and accommodating foreign DNA.
  • gRNA refers to a guide RNA of about 20 nt, which can be combined with target editing site DNA in a form of base complementation, so that the Cas9 protein can stably bind to the target site and perform base cutting.
  • the term "resistance screening" means that the recipient bacteria without antibiotic resistance genes cannot grow in the antibiotic-containing medium, and the recipient bacteria can only grow after the vector with antibiotic resistance genes enters the recipient bacteria.
  • the antibiotic resistance gene in the present invention can be a conventional vector resistance gene in the art, such as spectinomycin resistance gene, kanamycin resistance gene, ampicillin resistance gene or hygromycin resistance gene. For example, after transfecting a plasmid with a kanamycin resistance gene into a recipient bacteria, the recipient bacteria that originally did not carry the kanamycin resistance and the recipient bacteria that were transferred with the kanamycin resistance gene are in the same time.
  • Double resistance screening means that one or more vectors transferred into the recipient bacteria contain two antibiotic resistance genes, similarly, the transfer containing two antibiotic resistance genes can be screened on a medium containing two antibiotics Recipient bacteria for genes.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • clustered regularly spaced short Palindromic Repeats is a family of special DNA repeats that are widely present in the genomes of bacteria and archaea.
  • the gene sequence of CRISPR-Cas system consists of three parts, including tracrRNA, Cas gene, and CRISPR locus composed of repetitive sequence and spacer sequence.
  • the length of the repetitive sequence is about 21 ⁇ 48bp, which is a highly conserved sequence, and the length of the spacer sequence is about 26.
  • ⁇ 72bp usually considered to be derived from exogenous nucleic acids such as plasmids and phages, is the main element for identifying target genes; Cas gene encodes Cas protein with nuclease function, tracrRNA plays the role of connecting CRISPR locus transcription product crRNA and Cas protein.
  • the spacer sequence is transcribed into crRNA, crRNA and tracrRNA form a double-stranded secondary structure, that is, sgRNA, sgRNA and Cas protein form a complex, sgRNA specifically recognizes the gene site, and Cas protein acts as a nucleic acid Enzyme activity performs gene cleavage.
  • the CRISPR-Cas system that has been discovered so far includes three types, namely type I, type II and type III.
  • the type I system contains the characteristic Cas3 protein
  • the type III system contains the characteristic Cas10 protein.
  • CRISPR has a complex structure and includes multiple interacting Cas proteins. It is difficult to perform molecular biology operations at this stage, which limits its application in gene editing.
  • the type II system contains only one Cas9 protein, which has simple molecular operations and functions. The mechanism is basically clear, so the Type II CRISPR-Cas system is widely used.
  • the CRISPR-Cas type II system namely CRISPR-Cas9 technology, is mainly used in the present invention.
  • ZFNs Zinc-finger nucleases
  • Each zinc finger nuclease consists of two functional domains: a.) DNA binding domain: consists of a two-finger module chain, each module recognizes a unique hexamer (6bp) DNA sequence, double Finger modules are spliced together to form a zinc finger protein, each protein has a specificity of ⁇ 24bp; b.) DNA cleavage domain: It is composed of the nuclease domain of Fok I. When the DNA binding domain and the DNA cleavage domain are fused together, a highly specific "genomic scissors" is produced.
  • TALENs transcription activator-like effector nuclease technology
  • TAL effectors a natural protein secreted by plant bacteria. Recognize specific DNA base pairs. TAL effectors can be designed to recognize and bind all target DNA sequences. Adding a nuclease to the TAL effector generates TALENs. The TAL-effect nuclease can bind to DNA and cut the DNA strand at a specific site, thereby introducing new genetic material.
  • sequence Identity refers to the amount of identity between two sequences (for example, a query sequence and a reference sequence), and is generally expressed as a percentage.
  • sequence alignment is performed and gaps (if any) are introduced. If at a certain alignment position, the bases or amino acids in the two sequences are the same, it is considered that the two sequences are identical or matched at that position; if the bases or amino acids in the two sequences are different, it is considered that they are inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity.
  • the number of gaps and/or the length of the gaps are also taken into account.
  • the published alignment software BLAST available on the webpage ncbi.nlm.nih.gov
  • BLAST can be used to obtain the best sequence alignment and calculate the sequence between the two sequences by using the default settings consistency.
  • Genetically modified engineered bacteria such as TOP10F (Barstar) and JM108 (Barstar) can be used for the cloning of barnase gene-related plasmids, providing a fast and stable solution for barnase-related gene synthesis and plasmid construction.
  • the engineered bacteria constructed by the invention can be directly used for the construction of barnase gene-related plasmids without other auxiliary elements; and the cloning success rate is much higher than previous technical methods, greatly shortening the time and cost of gene synthesis and cloning.
  • Figure 1 is a design scheme of gRNA, gRNA-1, gRNA-2 and gRNA-3 respectively target the sequence of ⁇ 50bp downstream of the CyaA terminator codon on the E. coli genome;
  • Figure 2 shows the barstar expression element, which contains the EM7 promoter, Barstar coding sequence, rrnBT1 and T7Te terminator;
  • Figure 3 is a flow chart of the construction of a dual plasmid system.
  • the synthesized Donor sequence is cloned into the pTarget plasmid to obtain the Donor plasmid, which then forms a dual plasmid system with pCas9;
  • FIG. 4 is a flowchart of E. coli genome editing.
  • Donor plasmid and pCas9 plasmid are co-transfected into E. coli, Donor sequence is integrated into E. coli chromosomal DNA, and pCas9 plasmid is cultivated and eliminated;
  • Figure 5 is a plate diagram of a plasmid transformation experiment containing barnase gene
  • Figure 5A is a plate diagram of three plasmids containing barnase (pUC57-barnase, U9041-1, U9041-12) respectively transformed into TOP10F and TOP10F (Barstar) culture;
  • Figure 5B is a plate diagram of three plasmids containing barnase (pUC57-barnase, U9041-1, U9041-12) after being transformed into JM108 and JM108 (Barstar), respectively, in which TOP10F and JM108 are used as the transformation control group;
  • Figure 6 shows the electrophoresis diagram of the plasmid containing barnase.
  • the sequenced TOP10F Barstar#1 and #2, JM08 Barstar#1 and #2, digested with HindIII and XbaI, electrophoresis, lanes 1, 4, 7, 10, 12, 15, 18 and 21 are respectively 300ng plasmid before digestion; Lanes 2, 5, 8, 11, 13, 16, 19 and 22 are respectively 300ng (HindIII+XbaI) plasmid after digestion; Lanes 3, 6, 9 , 14, 17 and 20 are DNA Marker (M), and the corresponding band size is shown in the molecular weight marker on the right.
  • M DNA Marker
  • the present invention uses CRISPR-Cas9 technology to insert the Barstar gene into TOP10F (Invitrogen, C303003) and JM108 (ATCC, 47107 TM ) E. coli genomes, so as to realize Barnase gene-related plasmid cloning in these two modified E. coli.
  • the invention is realized by the following technical route: firstly, genetically transform two kinds of Escherichia coli and insert the barstar expression element; and then test the transformation effect of the plasmid containing the barnase gene in the transformed Escherichia coli.
  • gRNA-1 GCCGGAAAGCGAGGCTTATC (SEQ ID NO: 7)
  • gRNA-2 GCCGGATAAGCCTCGCTTTC (SEQ ID NO: 8)
  • gRNA-3 TTTCCGCTAAGATTGCATGC (SEQ ID NO: 9)
  • the pCas plasmid (from Jiang et.al, 2015, Applied and Environmental Microbiology) was electroporated into competent cells of TOP10F and JM108. Since pCas is a temperature-sensitive plasmid, the activation and culture temperature during the electroporation process needs to be set at 30°C.
  • the universal primer sequence is as follows:
  • Cas9-F actagccagcatccgtttacga (SEQ ID NO: 10)
  • Cas9-R tgcgcaaagtattgtccatgcc (SEQ ID NO: 11)
  • the positive cloned strains (pCas in TOP10F and pCas in JM108) obtained in the previous step can be divided on the Kanamycin (Kan) plate. After a single bacteria line is obtained, a single colony is picked and activated in LB liquid medium containing Kan overnight.
  • the cells were collected and centrifuged at 4000 rpm for 4 min, the supernatant was removed, and 100 ⁇ L was applied to a double antibody plate containing kanamycin and spectinomycin (Spectinomycin, Spec), and placed in a 30°C incubator for overnight culture.
  • kanamycin and spectinomycin Spectinomycin
  • PCR primers for the knocked-in target gene region perform PCR bacterial detection on a single colony on the plate, and screen positive clones.
  • Bacteria detection primers (F1+R2) are used to detect whether the target gene region on the genome is inserted; the primers (F1+R1) are used to detect whether Barstar is inserted into the target region of the genome.
  • R1 TTATGAAGTCTGAAAAGTGAGGAAA (SEQ ID NO: 13)
  • R2 TCTTCATCAACTACCTCCTAT (SEQ ID NO: 14)
  • Transformed TOP10F (Barstar) and JM108 (Barstar) E. coli cells refer to steps 2.2 to 2.4 of Example 1 to prepare transformed receptor cells.
  • Two test plasmids (U9041-1 and U9041-12, containing the Kan resistance gene, see SEQ ID NO: 4 and 5 for the full sequence) containing barnase (see SEQ ID NO: 3 for sequence information) and the control plasmid pUC57- barnase (see SEQ ID NO: 6 for sequence information), respectively transfer to TOP10F and JM108, as well as the modified TOP10F (Barstar) and JM108 (Barstar) receptor cells, overnight on the (LB+Kan) plate at 37°C nourish.
  • SEQ ID NO.1 barstar gene
  • SEQ ID NO. 2 barstar expression element sequence
  • SEQ ID NO.3 barnase gene
  • SEQ ID NO. 4 Full sequence of U9041-1 plasmid
  • the bold and underlined part represents the barnase gene
  • SEQ ID NO.5 Full sequence of U9041-12 plasmid
  • the bold and underlined part represents the barnase gene
  • SEQ ID NO.6 Full sequence of pUC57-barnase plasmid
  • the bold and underlined part represents the barnase gene

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Abstract

提供了含barstar基因的工程菌及其在barnase基因克隆中的应用，其中，所述工程菌的染色体基因组中包含barstar基因，所述工程菌能用于克隆barnase基因。还提供了所述barnase基因的克隆方法，包括将包含barnase基因的质粒转化到上述的工程菌中培养扩增。

Description

含barstar基因的工程菌及其在barnase基因克隆中的应用 技术领域
本发明属于基因工程领域，具体涉及含barstar基因的工程菌及其构建方法，以及所述工程菌在核糖核酸酶barnase基因克隆中的应用。
背景技术
Barnase是解淀粉芽孢杆菌(Bacillus amyloliquefaciens)产生的一种12kD的胞外小分子RNA酶，它可以降解细胞中的RNA，具有强烈的毒性。在解淀粉芽孢杆菌基因组中也含有barnase的特异性拮抗基因barstar，其产物(10kD)能与Barnase以1:1的比例特异性结合，形成高度稳定的复合物,从而使细菌表达的Barnase失去酶活性 [1]。利用它在特异细胞中的表达将造成细胞的死亡这一特点，科学家用植物育性或抗病相关启动子去调控barnase在细胞中活性，从而实现了植物的雄性不育以及抗病性的提高。因此该基因在农业生产上具有广泛的用途 [2]。此外，Barnase还具有其它许多重要的生物学功能，如控制肿瘤血管生成，杀灭肿瘤细胞及抑制病毒(包括HIV病毒)的复制等 [3]。以上特点使barnase成为质粒序列中一种常见的元件，应用于各种实验中。
Barnase基因在大肠杆菌中的克隆常见方法有以下几种。一种是采用PCR直接克隆，根据barnase编码区序列设计引物，直接从解淀粉芽孢杆菌的总DNA扩增barnase序列，然后克隆到载体中 [4,5]。该方法的缺点是由于Barnase的毒性作用，得到的基因总是带有突变，得到正确序列的概率极低。第二种是保护性克隆，将barnase基因置于barstar基因的保护下进行克隆。通常是将barnase与barstar克隆于同一质粒载体上 [6]。该方法的缺点是最终的质粒上有额外的barstar基因，影响后续的下游应用。其三是在barnase基因内部插入一个内含子序列，使得在大肠杆菌里面表达的barnase含有额外序列而失去毒性，而在真核细胞内去除内含子序列后恢复活性 [7]。该方法的缺点是没有真核细胞通用的内含子序列。
1.Hartley,R.W.,Barnase and barstar.Expression of its cloned inhibitor permits expression of a cloned ribonuclease.J Mol Biol,1988.202(4):p.913-5.
2.Mariani,C.,et al.,A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants.Nature,1992.357(6377):p.384-387.
3.Leuchtenberger,S.,et al.,Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells.Nucleic Acids Res,2001.29(16):p.E76.
4.Paddon,C.J.and R.W.Hartley,Expression of Bacillus amyloliquefaciens extracellular ribonuclease(barnase)in Escherichia coli following an inactivating mutation.Gene,1987.53(1):p.11-9.
5.刘玉乐,et al.,核酸酶BN及SN基因的克隆和序列分析.微生物学通报,1992.4:p.200-203.
6.Deyev,S.,Waibel,R.,Lebedenko,E.et al.Design of multivalent complexes using the barnase·barstar module.Nat Biotechnol 21,1486–1492(2003).
7.孙路路,内含肽介导被拆分的Barnase毒蛋白功能重建.西南大学硕士论文.2012.
发明内容
由于前面所述的barnase克隆技术存在成功率低，无法满足下游应用，缺乏通用内含子序列等缺点，无法实现barnase基因克隆的所有情景。本发明通过CRISPR-Cas9技术对常规工程菌进行遗传改造。在所述工程菌的基因组上插入barstar基因的表达元件，使得基因组上表达的Barstar可以中和质粒上表达的Barnase毒性作用，从而能够顺利完成基因合成，质粒构建和质粒抽提。
本发明一方面提供了一种工程菌，其特征在于，所述工程菌的染色体基因组中包含barstar基因，所述工程菌能用于克隆barnase基因。
本发明中所述barstar基因包含SEQ ID NO：1所示核苷酸序列，或与SEQ ID NO：1所示核苷酸序列至少80％一致性的序列。在一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少80％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在另一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少91％一致性的序列。在一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少93％一致性的序列。在另一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少95％一致性的序列。在一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少97％一致性的序列。在另一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少99％一致性的序列。在一些实施方案中，所述barstar基因包含SEQ ID NO：1所示核苷酸序列。在一个具体实施方案中，所述barstar基因的核苷酸序列如SEQ ID NO：1所示。
在一些实施方案中，所述barstar基因来源于解淀粉芽孢杆菌。
在一些实施方案中，所述工程菌染色体基因组包括含barstar基因的表达元件。所述barstar基因表达元件包括启动子和终止子。在一些实施方案中，所述barstar基因表达元件包含持续性表达的启动子。在一些实施方案中，所述barstar基因表达元件包含的启动子选自EM7、recA、trp、araBAD、TEF1、GAL1、GAL10、lac、psbA、T7或tacP启动子中的一种或多种；所述barstar基因表达元件包含的终止子选自MrrnB T1、T7Te、rrnBT1、rrnBT2、rrnB1或rrnB2终止子中的一种或多种。优选地，所述启动子为EM7启动子，终止子为MrrnB T1终止子和T7Te终止子。
在一些实施方案中，所述barstar基因表达元件包含SEQ ID NO:2所示核苷酸序列，或与SEQ ID NO:2所示核苷酸序列至少80％一致性的序列。在另一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少80％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少91％一致性的序列。在另一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少93％一致性的序列。在一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少95％一致性的序列。在另一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少97％一致性的序列。在一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少99％一致性的序列。在 另一些实施方案中，所述barstar基因表达元件包含SEQ ID NO:2所示核苷酸序列。在一个具体实施方案中，所述barstar基因表达元件的核苷酸序列如SEQ ID NO:2所示。
在一些实施方案中，所述工程菌选自细菌或真菌；优选地，所述细菌选自大肠杆菌或枯草芽孢杆菌，所述真菌选自酵母菌；更优选地，所述大肠杆菌选自TOP10F、JM108、DH5a、stbl3、JM109、DH10b、EPI300或EPI400。在一些实施方案中，所述工程菌为大肠杆菌。在另一些实施方案中，所述工程菌为枯草芽孢杆菌。在一些实施方案中，所述大肠杆菌工程菌选自TOP10F、JM108、DH5a、stbl3、JM109、DH10b、EPI300或EPI400。在一些具体实施方案中，所述工程菌选自TOP10F或者JM108。在一个实施方案中，所述工程菌为TOP10F。在另一个实施方案中，所述工程菌为JM108。
在一些实施方案中，所述barstar基因克隆到大肠杆菌工程菌染色体中lacZ，recA，araD，dam，galE，galU，malA，ompT，tonA，rha或者CyaA基因的下游；优选地克隆到大肠杆菌工程菌染色体中CyaA基因的下游。在一些实施方案中，所述barstar基因克隆到大肠杆菌工程菌染色体中CyaA基因的下游。
在一些实施方案中，所述barstar基因表达元件克隆到大肠杆菌工程菌染色体中lacZ，recA，araD，dam，galE，galU，malA，ompT，tonA，rha或者CyaA基因的下游；优选地克隆到大肠杆菌工程菌染色体中CyaA基因的下游。在另一些实施方案中，所述barstar基因表达元件克隆到大肠杆菌工程菌染色体中CyaA基因的下游。
本发明另一方面提供了一种制备工程菌的方法，其特征在于，通过基因编辑技术将barstar基因克隆到工程菌的染色体基因组中。
本发明提供的制备方法中，所述barstar基因包含SEQ ID NO：1所示核苷酸序列，或与SEQ ID NO：1所示核苷酸序列至少80％一致性的序列。在一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少80％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在另一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少91％一致性的序列。在一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少93％一致性的序列。在另一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少95％一致性的序列。在一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少97％一致性的序列。在另一些实施方案中，所述barstar基因包含与SEQ ID NO：1所示核苷酸序列至少99％一致性的序列。在一些实施方案中，所述barstar基因包含SEQ ID NO：1所示核苷酸序列。在一个具体实施方案中，所述barstar基因的核苷酸序列如SEQ ID NO：1所示。
在一些实施方案中，所述工程菌染色体基因组中还包括基因编辑技术克隆的含barstar基因的表达元件，所述barstar基因表达元件包含启动子和终止子。在一些实施方案中，所述barstar基因表达元件包含的启动子选自EM7、recA、trp、araBAD、TEF1、GAL1、GAL10、lac、psbA、T7或tacP启动子中的一种或多种；所述barstar基因表达元件包含的终止子选自MrrnB T1、T7Te、rrnBT1、rrnBT2、rrnB1或rrnB2终止子中的一种或多种。优选地，所述启动子为EM7启动子，终止子为MrrnB T1终止子和T7Te终止子。
在一些实施方案中，所述barstar基因表达元件包含SEQ ID NO:2所示核苷酸序列，或与SEQ ID NO:2所示核苷酸序列至少80％一致性的序列。在另一些实施方案中，所述 barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少80％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少91％一致性的序列。在另一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少93％一致性的序列。在一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少95％一致性的序列。在另一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少97％一致性的序列。在一些实施方案中，所述barstar基因表达元件包含与SEQ ID NO:2所示核苷酸序列至少99％一致性的序列。在另一些实施方案中，所述barstar基因表达元件包含SEQ ID NO:2所示核苷酸序列。在一个具体实施方案中，所述barstar基因表达元件的核苷酸序列如SEQ ID NO:2所示。
本发明提供的制备方法中，所述基因编辑技术选自CRISPR-Cas9技术、锌指核酸酶技术或者转录激活因子样效应物核酸酶技术。优选地，所述基因编辑技术为CRISPR-Cas9技术。
本发明提供的制备方法中，所述基因编辑技术为CRISPR-Cas9技术，包括如下步骤：
(1)合成左右同源臂序列和barstar基因表达元件序列，将合成的序列插入到含gRNA序列的pTarget质粒，其中，所述左右同源臂序列分别是barstar基因克隆至所述工程菌基因靶序列的左侧和右侧的一段序列；
(2)步骤(1)得到的质粒与pCas9质粒共转染至工程菌细胞中，培养孵育；
(3)步骤(2)培养的工程菌通过抗性筛选得到barstar基因整合到工程菌染色体基因组中的菌株。
在本发明所述方法中，所述工程菌为大肠杆菌或者枯草芽孢杆菌；优选地，所述工程菌选自TOP10F、JM108、DH5a、stbl3、JM109、DH10b、EPI300或EPI400；更优选地，所述工程菌为TOP10F或者JM108。在一些实施方案中，所述工程菌为大肠杆菌。在另一些实施方案中，所述工程菌为枯草芽孢杆菌。在一些实施方案中，所述大肠杆菌工程菌选自TOP10F、JM108、DH5a、stbl3、JM109、DH10b、EPI300或EPI400。在一些具体实施方案中，所述工程菌选自TOP10F或者JM108。在一个实施方案中，所述工程菌为TOP10F。在另一个实施方案中，所述工程菌为JM108。在一些实施方案中，所述barstar基因克隆到大肠杆菌工程菌染色体中lacZ，recA，araD，dam，galE，galU，malA，ompT，tonA，rha或者CyaA基因的下游。在一些实施方案中，所述barstar基因克隆到大肠杆菌工程菌染色体中CyaA基因的下游。在另一些实施方案中，所述barstar基因表达元件克隆到大肠杆菌工程菌染色体中lacZ，recA，araD，dam，galE，galU，malA，ompT，tonA，rha或者CyaA基因的下游。在另一些实施方案中，所述barstar基因表达元件克隆到大肠杆菌工程菌染色体中CyaA基因的下游。在一些实施方案中，所述barstar基因克隆至大肠杆菌工程菌的基因靶序列选自lacZ，recA，araD，dam，galE，galU，malA，ompT，tonA，rha或者CyaA基因的下游序列；优选地克隆至大肠杆菌工程菌基因的靶序列为CyaA基因的下游序列。
在一些实施方案中，所述步骤(1)中gRNA是根据barstar基因克隆至工程菌的基因位点设计的。其中，所述步骤(1)中gRNA的靶向序列(barstar基因克隆至所述工程菌基因靶序列)为位于大肠杆菌工程菌基因组上CyaA基因终止密码子下游的1-200bp序列。优选地，所述步骤(1)中gRNA的靶向序列为大肠杆菌工程菌基因组CyaA基因终止密码子下 游的1-100bp的序列。更优选地，所述步骤(1)中gRNA的靶向序列为大肠杆菌工程菌基因组上CyaA基因终止密码子下游的1-50bp序列。在一些具体实施方案中，所述步骤(1)中gRNA的靶向序列为大肠杆菌工程菌基因组上CyaA基因终止密码子下游的10-30bp序列。其中，所述gRNA的靶向序列3’端相邻的序列为NGG。在一些实施方案中，所述步骤(1)中gRNA的序列长度为10-30bp，优选地，所述gRNA的序列长度为20bp。在一些实施方案中，所述gRNA包括至少1条序列，优选为至少2条序列，更优选为3条序列。在另一些实施方案中，所述gRNA包含与SEQ ID NO:7、8或/和9所示核苷酸序列至少80％一致性的序列。在一些实施方案中，所述gRNA包含与SEQ ID NO:7、8或/和9所示核苷酸序列至少80％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述gRNA包含SEQ ID NO:7、8和9所示核苷酸序列。
在一些实施方案中，所述步骤(1)中包含gRNA序列的pTarget质粒是以空载体pTarget质粒为模板进行突变、扩增得到的。在另一些实施方案中，所述步骤(1)中左右同源臂序列是通过扩增所述工程菌的靶序列左右两侧50-500bp序列得到的。在一些实施方案中，所述步骤(1)中合成的左右同源序列与barstar基因表达元件序列扩增融合后，再插入到含gRNA序列的pTarget质粒。具体地，所述步骤(1)包括：a.以空载体pTarget质粒为模板进行突变、扩增得到含有gRNA的pTarget质粒；b.根据gRNA的靶序列的左右两边选择50-500bp的序列进行PCR扩增，得到同源重组的左右同源臂序列；c.把同源臂序列和barstar表达元件序列进行融合，再插入到pTarget-gRNA质粒中，测序验证后，得到含有gRNA和barstar表达元件序列的pTarget质粒。更具体地，所述步骤(1)包括以空载体pTarget质粒为模板进行点突变扩增得到含有gRNA的pTarget质粒，再在工程菌的基因组上，gRNA的靶序列的左右两边选择500bp的序列进行PCR扩增，得到同源重组的左右同源臂序列；把同源臂序列和barstar表达元件序列通过PCR进行融合，得到的产物通过EcoRI-HindIII酶切位点插入到pTarget-sgRNA质粒中，测序验证后，得到含有gRNA和barstar表达元件序列的pTarget质粒。
在一些实施方案中，所述步骤(1)中得到的质粒和步骤(2)的pCas9质粒分别包含至少一种抗性基因。在一些具体实施方案中，所述包含gRNA的质粒和pCas9质粒的抗性基因分别选自壮观霉素抗性基因、卡那霉素抗性基因、氯霉素抗性基因或氨苄霉素抗性基因。在一个具体实施方案中，所述包含gRNA的质粒包含壮观霉素抗性基因，所述pCas9质粒包含卡那霉素抗性基因。
在一些实施方案中，所述步骤(2)包括在30℃下，先将具有第一抗性的pCas9质粒电转入工程菌中，接着在含有第一抗性的培养基中筛选得到成功转入pCas9质粒的菌株，在含有第一抗性的液体培养基中过夜活化菌株，再按照1：100-1000(V:V)的比例转接至含有第一抗性的液体培养基中，培养一段时间后，加入1：1-100(V:V)的1ml阿拉伯糖诱导细胞继续生长，直至培养到OD600为0.4-0.6，将菌液在4℃下离心沉淀，去上清，用1％-80％甘油洗涤、并重悬浮菌株，后加入步骤(1)得到的含有第二抗性抗性的pTarget质粒进行电转，电转后加入液体培养基进行活化；再将菌株在含有第一抗性和第二抗性的培养基中培养过夜，最后进行PCR菌检，筛选出基因组上导入了barstar基因的阳性菌株。其中所述第一抗性和第二抗性选自壮观霉素抗性基因、卡那霉素抗性基因、氯霉素抗性基因或氨苄霉素抗性基因。
在一些实施方案中，所述步骤(2)包括在30℃下，先将具有Kan抗性的pCas9质粒电转入工程菌中，接着在含有Kan的培养基中筛选得到成功转入pCas9质粒的菌株，在含有Kan的液体培养基中过夜活化菌株，再按照1：200-800(V:V)的比例转接至含有Kan的液体培养基中，培养一段时间后，加入1：100(V:V)的1ml阿拉伯糖诱导细胞继续生长，直至培养到OD600为0.4-0.6，将菌液在4℃下离心沉淀，去上清，用10％甘油洗涤、并重悬浮菌株，后加入步骤(1)得到的含有Spec抗性的pTarget质粒进行电转，电转后加入液体培养基进行活化；再将菌株在含有Kan和Spec的培养基中培养过夜，最后进行PCR菌检，筛选出基因组上导入了barstar基因的阳性菌株。
在一些实施方案中，所述步骤(2)包括在温度30℃下，先将具有Kan抗性的pCas9质粒电转入大肠杆菌中，接着在含有Kan的培养基中筛选得到成功转入pCas9质粒的菌株，在含有Kan的液体培养基中过夜活化菌株，再按照1：400(V:V)的比例转接至含有Kan的液体培养基中，培养一段时间后，加入1：100(V:V)的1ml阿拉伯糖诱导细胞继续生长，直至培养到OD600为0.4-0.6，将菌液在4℃下离心沉淀，去上清，用10％甘油洗涤、并重悬浮菌株，后加入步骤(1)得到的含有Spec抗性的pTarget质粒进行电转，电转后加入液体培养基进行活化；再将菌株在含有Kan和Spec的培养基中培养过夜，最后进行PCR菌检，筛选出基因组上导入了barstar基因的阳性菌株。
在一些实施方案中，所述步骤(2)包括在温度30℃下，先将具有Kan抗性的pCas9质粒电转入TOP10F或者JM108中，接着在含有Kan的培养基中筛选得到成功转入pCas9质粒的菌株，在含有Kan的液体培养基中过夜活化菌株，再按照1：400(V:V)的比例转接至含有Kan的液体培养基中，培养一段时间后，加入1：100(V:V)的1ml阿拉伯糖诱导细胞继续生长，直至培养到OD 600为0.4-0.6，将菌液在4℃下离心沉淀，去上清，用10％甘油洗涤、并重悬浮菌株，后加入步骤(1)得到的含有Spec抗性的pTarget质粒进行电转，电转后加入液体培养基进行活化；再将菌株在含有Kan和Spec的培养基中培养过夜，最后进行PCR菌检，筛选出基因组上导入了barstar基因的阳性菌株。
在一些实施方案中，所述步骤(3)中所述抗性筛选是指通过双抗性培养基筛选出barstar基因整合到染色体基因组中的工程菌株。在一些实施方案中，所述步骤(3)中所述抗性筛选是通过含有Kan或者Spec中一种或两种抗性的培养基筛选出barstar基因整合到染色体基因组中的工程菌株。
在一些实施方案中，所述步骤(3)包括将步骤(2)得到的阳性工程菌，挑取单菌落接种到含有第一抗性的培养基中，加入0.1-1mM IPTG诱导菌株表达，再将菌株分别接种在含有第一抗性和含有第二抗性的两种培养基中，30℃培养后，挑选出在含有第二抗性的培养基中不能生长，而在含有第一抗性正常生长的菌株，即为消除了pTarget质粒的菌株，再将菌株多次接种于无抗性的培养基中，37℃培养后，然后再将菌株分别接种到含有第一抗性和含有第二抗性的两种培养基，当菌株在这两种培养基中分别都不生长时，即得到消除pCas9和pTarget的工程菌株。
在一些实施方案中，所述步骤(3)包括将步骤(2)得到的阳性工程菌，挑取单菌落接种到含有Kan的培养基中，加入0.1-0.8mM IPTG诱导菌株表达，再将菌株分别接种在含有Spec和含有Kan的两种培养基中，30℃培养后，挑选出在含有Spec的培养基中不能生长，而在含有Kan的培养基正常生长的菌株，即为消除了pTarget质粒的菌株，再将菌株多次接 种于无抗性的液体培养基中，37℃培养后，然后再将菌株分别接种到含有Kan和含有Spec的两种培养基中，当菌株在两种培养基中分别都不生长时，即得到消除pCas9和pTarget的工程菌株。
在一些实施方案中，所述步骤(3)包括将步骤(2)得到的大肠杆菌，挑取单菌落接种到含有Kan的LB培养基中，加入0.5mM IPTG诱导菌株表达，再将菌液分别接种在含有Spec和含有Kan的两种LB培养基中，30℃培养后，挑选出在含有Spec的培养基中不能生长，而在含有Kan正常生长的菌株，即为消除了pTarget质粒的菌株，再将菌株多次接种于无抗性的LB培养基中，37℃培养后，然后再将菌株分别接种到含有Kan和含有Spec的两种LB培养基，当菌株在两种培养基中分别都不生长时，即得到消除pCas9和pTarget的工程菌株。
在一些实施方案中，所述步骤(3)，优选地，包括将步骤(2)得到的TOP10F或者JM108，挑取单菌落接种到含有Kan的LB培养基中，加入0.5mM IPTG诱导菌株表达，再将菌株分别在含有Spec和含有Kan的两种LB培养基中，30℃培养后，挑选出在含有Spec的培养基中不能生长，而在含有Kan正常生长的菌株，即为消除了pTarget质粒的菌株，再将菌株接种于无抗性的LB液体培养基中，37℃培养后，然后再将菌株分别接种到含有Kan和含有Spec的两种LB培养基，当菌株在两种培养基中分别都不生长，即得到消除pCas9和pTarget的工程菌株。
本发明又一方面提供了一种克隆barnase基因的方法，其特征在于，所述方法包括将包含barnase基因的质粒转染到上述工程菌中培养扩增。
本发明还提供了一种克隆barnase基因的方法，其特征在于，所述方法包括将包含barnase基因的质粒转化到上述方法制备的工程菌中培养扩增。
上述克隆barnase基因的方法中，进一步包括通过双抗性培养基筛选出转化barnase基因的工程菌。在一些实施方案中，通过Kan抗性和/或Spe抗性培养基筛选得到所述转化barnase基因的工程菌的。优选地，通过Kan抗性和Spe抗性培养基筛选得到所述转化barnase基因的工程菌。
在一些实施方案中，所述方法还包括对扩增培养的工程菌进行质粒提取。
在另一些实施方案中，所述方法进一步包括将提取到的质粒进行序列验证。
在一些实施方案中，所述包含barnase基因的质粒选自原核表达载体pET、pGEX系列、酵母载体pRS、pESC系列、植物载体pCAMBIA系列、真核表达载体pcDNA3.1系列、pUC57、U9041-1或者U9041-12。在一些优选实施方案中，所述包含barnase基因的质粒pUC57、U9041-1或者U9041-12。
术语解释
本发明中所述“工程菌”可以是任何原核细胞或真核细胞的微生物，原核细胞微生物可以是细菌，包括但不限于大肠杆菌、枯草芽孢杆菌，真核细胞微生物包括但不限于酵母菌、曲霉菌。本发明中的一些实施方案中，所述工程菌为大肠杆菌或者枯草芽孢杆菌。其中，大肠杆菌可选自TOP10F、JM108、DH5a、stbl3、JM109、DH10b、EPI300或EPI400。在本发明的一个具体实施方案中，所述工程菌为TOP10F或者JM108。
术语“Donor序列”或“供体序列”是指在同源重组中，人工构建一段序列，该序列含有 与靶向位点上下游一定长度(比如50-500bp)的同源序列，从而使靶向位点通过同源重组的方式插入或缺失一段序列。
本发明中“同源臂序列”是指一段与靶向序列上下游具有相同序列的DNA片段，用于识别靶向序列并发生同源重组的区域，一般长度为50-500bp。
术语“载体”在本文中用以指能够转移或输送另一个核酸分子的核酸分子。转移后的核酸通常连接到载体核酸分子，例如插入载体核酸分子中。载体可以包括在细胞中指导自主复制的序列，或可以包括足以允许整合到宿主细胞DNA中的序列。有用的载体包括例如质粒(例如DNA质粒或RNA质粒)、转座子、粘粒、细菌人工染色体以及病毒载体。
术语“转染”指多核苷酸或其他生物活性化合物从细胞外转移到细胞内，使得该多核苷酸或生物活性化合物是功能性的。用于在体外将多核苷酸递送到细胞的转染试剂的实例包括但不限于：脂质体、脂类、多胺、磷酸钙沉淀、组蛋白、聚氮丙啶和两性聚电解质复合物和这些的组合。许多体外转染试剂是阳离子的，其允许所述试剂与带负电荷的核酸通过静电相互作用结合或形成复合体。
“共转染”是指两个或多个质粒一同转入同一个感受态细胞，可采用本领域常用的转染方法，包括电转、化学转染等方法将两个或多个质粒同时转入同一个感受态的细胞。本发明中优选为电转方法，将两个或多个质粒转入同一个感受态细胞中。
术语“感受态细胞”是指通过理化方法诱导细胞，吸收周围环境中的DNA分子，使其处于最适摄取和容纳外来DNA的生理状态。
本发明中gRNA是指一段20nt左右的向导RNA，可以通过碱基互补的形式与靶向编辑位点DNA进行结合，从而使Cas9蛋白稳定结合到靶向位点并进行碱基切割。
术语“抗性筛选”是指不带有抗生素抗性基因的受体菌不能在含有抗生素的培养基中生长，只有带有抗生素抗性基因的载体进入受体菌后，受体菌才能生长。本发明中抗生素抗性基因可以是本领域常规的载体抗性基因，如壮观霉素抗性基因、卡那霉素抗性基因、氨苄霉素抗性基因或潮霉素抗性基因等。如将带有卡那霉素抗性基因的质粒转染如受体菌后，原本不带卡那霉素抗性的受体菌和转入卡那霉素抗性基因的受体菌同时在含卡那霉素的培养基上培养，只有含卡那霉素抗性基因的受体菌能生长，从而被筛选出来。“双抗性筛选”是指转入受体菌的一个或多个载体包含两种抗生素抗性基因，类似的，在含两种抗生素的培养基上能筛选出转入包含两种抗生素抗性基因的受体菌。
术语“CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)”或“成簇规律间隔短回文重复序列”，是一个广泛存在于细菌和古生菌基因组中的特殊DNA重复序列家族。CRISPR-Cas系统基因序列由三部分组成，包括tracrRNA、Cas基因以及由重复序列和间隔序列组成的CRISPR基因座，其中重复序列长度约为21～48bp，为高度保守序列，间隔序列长度约为26～72bp，通常认为来自质粒、噬菌体等外源核酸，为识别靶基因的主要元件；Cas基因编码具有核酸酶功能的Cas蛋白，tracrRNA起到连接CRISPR基因座转录产物crRNA和Cas蛋白的作用。在CRISPR-Cas系统作用过程中，间隔序列被转录为crRNA，crRNA与tracrRNA形成双链二级结构，即sgRNA，sgRNA与Cas蛋白形成复合体，由sgRNA特异性识别基因位点，Cas蛋白发挥核酸酶活性进行基因切割。基于该原理，设计不同的间隔序列，即可对特定位点的基因进行敲除、插入、改造、修复等基因编辑操作。目前已发现的CRISPR-Cas系统包括三类，即Ⅰ型、Ⅱ型和Ⅲ型，其中Ⅰ型系统中包含特征性Cas3蛋白、Ⅲ型系统中包含特征性Cas10蛋白，但这两种系统中的CRISPR结构复杂， 均包括多种相互作用的Cas蛋白，现阶段难以进行分子生物学操作，限制了其在基因编辑中的应用，而Ⅱ型系统中仅含有一种Cas9蛋白，分子操作简单并作用机制已基本明确，故Ⅱ型CRISPR-Cas系统应用较为广泛。有研究表明通过利用Cas9的一种突变形式(只切割单链DNA)和两个紧挨着的向导RNA，有可能大幅提高系统的切割位点保真性。本发明中主要应用的是CRISPR-Cas Ⅱ型系统，即CRISPR-Cas9技术。
术语“锌指核酸酶(Zinc-finger nucleases,ZFNs)”，是人工改造的限制酶，通过融合锌指结构的结合DNA结构域和分解DNA结构域而成。“锌指核酸酶技术”可通过基因工程改造锌指结构域使锌指核酸酶针对复杂基因组里的特定DNA顺序，借助内源DNA的修复机制，锌指核酸酶可以精确改变高等动物的基因组。每个锌指核酸酶(ZFN)由两个功能结构域组成：a.)DNA结合结构域：由一个双指模块链组成，每个模块识别一个独特的六聚体(6bp)DNA序列，双指模块拼接在一起形成一个锌指蛋白，每个蛋白具有≥24bp的特异性；b.)DNA切割结构域：由Fok I的核酸酶结构域组成。当DNA结合结构域和DNA切割结构域融合在一起时，产生高度特异性的“基因组剪刀”。
术语“转录激活因子样效应物核酸酶技术(TALENs)”是一种可靶向修饰特异DNA序列的酶，它借助于TAL(transcription activator-like)效应子一种由植物细菌分泌的天然蛋白来识别特异性DNA碱基对。TAL效应子可被设计识别和结合所有的目的DNA序列。对TAL效应子附加一个核酸酶就生成了TALENs。TAL效应核酸酶可与DNA结合并在特异位点对DNA链进行切割，从而导入新的遗传物质。
提及序列时，术语“序列一致性(Sequence Identity)”(也称为“序列同一性”)指两序列(例如查询序列和参照序列)之间一致性程度的量，一般以百分比表示。通常，在计算两序列之间的一致性百分比之前，先进行序列比对(Alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置，两序列中的碱基或氨基酸相同，则认为两序列在该位置一致或匹配；两序列中的碱基或氨基酸不同，则认为在该位置不一致或错配。在一些算法中，用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中，还将缺口数量和/或缺口长度考虑在内。出于本发明的目的，可以采用公开的比对软件BLAST(可在网页ncbi.nlm.nih.gov找到)，通过使用缺省设置来获得最佳序列比对并计算出两序列之间的序列一致性。
有益技术效果
遗传改造后的工程菌，如TOP10F(Barstar)和JM108(Barstar)可用于barnase基因相关质粒的克隆，为barnase相关基因合成和质粒构建提供了快速和稳定的解决方案。本发明构建的工程菌可以直接用于barnase基因相关质粒构建，无需借助其它辅助元件；且克隆成功率远远高于以前的技术方法，大大缩短基因合成和克隆的时间和成本。
附图说明
图1为gRNA的设计方案，gRNA-1、gRNA-2及gRNA-3分别靶向大肠杆菌基因组上CyaA终止子密码子下游～50bp序列；
图2为barstar表达元件，包含有EM7启动子，Barstar的编码序列，rrnBT1和T7Te终止子；
图3为双质粒系统构建的流程图，合成的Donor序列克隆到pTarget质粒里面得到Donor质粒，然后和pCas9组成双质粒系；
图4为大肠杆菌基因组编辑的流程图，Donor质粒和pCas9质粒共转染大肠杆菌，Donor序列整合到大肠杆菌染色体DNA中，培养消除pCas9质粒；
图5为含有barnase基因质粒转化实验的平板图，图5A为含有barnase的三个质粒(pUC57-barnase，U9041-1，U9041-12)分别转化到TOP10F和TOP10F(Barstar)培养后的平板图；
图5B为含有barnase的三个质粒(pUC57-barnase，U9041-1，U9041-12)分别转化到JM108和JM108(Barstar)培养后的平板图，其中，TOP10F和JM108作为转化对照组；
图6为含有barnase质粒酶切验证的电泳图，测序正确的TOP10F Barstar#1和#2，JM08 Barstar#1和#2，用HindIII和XbaI酶切后电泳，泳道1，4，7，10，12，15，18和21分别是300ng酶切前质粒；泳道2，5，8，11，13，16，19和22分别是300ng(HindIII+XbaI)酶切后质粒；泳道3，6，9，14，17和20是DNA Marker(M)，对应条带大小见右侧分子量标记。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
本发明用CRISPR-Cas9技术把Barstar基因插入到TOP10F(Invitrogen,C303003)和JM108(ATCC,47107 TM)大肠杆菌基因组中，实现在这2种改造后的大肠杆菌中进行Barnase基因相关的质粒克隆。本发明通过以下技术路线实现：首先对两种大肠杆菌进行遗传改造，插入barstar表达元件；然后在改造后的大肠杆菌中测试含有barnase基因的质粒转化制效果。
实施例1菌种改造
1质粒构建
1.1在要插入的大肠杆菌基因组CyaA基因终止密码子下游(10-30bp以内)，选择3个20bp的gRNA靶序列，这3条gRNA靶向大肠杆菌基因组上CyaA终止子密码子下游～50bp序列，其中靶序列3’端相邻的序列为NGG，如图1所示。以空载体pTarget质粒(来源于Jiang et.al,2015,Applied and Environmental Microbiology)为模板进行点突变扩增，得到正确的pTarget-sgRNA质粒(其中含有壮观霉素(Spectinomycin，Spec)抗性基因)。
gRNA-1：GCCGGAAAGCGAGGCTTATC(SEQ ID NO:7)
gRNA-2：GCCGGATAAGCCTCGCTTTC(SEQ ID NO:8)
gRNA-3：TTTCCGCTAAGATTGCATGC(SEQ ID NO:9)
1.2分别在大肠杆菌基因组上的gRNA靶序列左右选择约500bp的序列进行PCR扩增，得到同源重组的左右同源臂序列；合成barstar表达元件序列，包含EM7启动子序列，barstar的编码序列，MrrnB T1和T7Te终止子序列，见图2。
1.3把同源臂序列和barstar表达元件序列通过overlapping PCR进行融合，得到的全长PCR产物通过EcoRI-HindIII酶切位点插入到pTarget-sgRNA质粒中，Sanger测序验证后，得到含有gRNA和donor(供体)序列(即同源臂序列+barstar表达元件序列)的pTarget-sgRNA-donor质粒。
2 pCas9感受态细胞制备和质粒共转
1.1将pCas质粒(来源于Jiang et.al,2015,Applied and Environmental Microbiology)电转至TOP10F和JM108的感受态细胞中。由于pCas是温敏型质粒，电转过程中的活化和培养温度需要设置在30℃。用通用引物Cas9-F和Cas9-R，对涂板后的克隆进行PCR菌检，得到阳性克隆后保菌用于下一步。通用引物序列如下：
Cas9-F:actagccagcatccgtttacga(SEQ ID NO:10)
Cas9-R:tgcgcaaagtattgtccatgcc(SEQ ID NO:11)
2.2由于pCas质粒中含有卡那霉素(Kanamycin，Kan)抗性基因，可以将上一步得到的阳性克隆菌株(pCas in TOP10F和pCas in JM108)分别在卡那霉素(Kanamycin，Kan)平板划线获得单菌落后，挑取单菌落在含有Kan的LB液体培养基中过夜活化。
2.2将过夜培养的菌按照1:400(V:V)的比例转接至新的15ml离心管，管内有4ml(LB+Kan)培养液，放置摇床培养(30℃，200rpm)。
2.3培养约2-3小时，在细胞浓度OD 600约为0.2左右时取出，加1:100(V:V)的1mL阿拉伯糖诱导细胞继续生长，直至细胞浓度OD 600约为0.5左右，将单管放冰上预冷即可准备做感受态(提前预冷离心机调至4℃)，转移至1.5ml离心管中，4000rpm离心4min，去除上清。
2.4用10％甘油清洗三次后，用100μL的10％甘油将其悬浮；加入200ng pTarget-sgRNA-donor质粒并混匀(全程冰上操作)。
2.5将BioRad电转仪(Bio-Rad Gene Pulser,165-2660)调到E2模式(KV：2.49V)，电击杯提前预冷，将混有产物的感受态细胞缓缓加入避免气泡，电转后加入LB液体培养基活化30℃，1h。
2.6活化后收集菌体4000rpm离心4min，去上清，留100μL涂布在含有卡那霉素和壮观霉素(Spectinomycin，Spec)的双抗平板，放置于30℃培养箱培养过夜。
2.7针对被敲入的目的基因区域设计PCR引物，对平板上的单菌落进行PCR菌检，筛选阳性克隆。菌检引物(F1+R2)用于检测基因组上目的基因区域是否有插入；引物(F1+R1)用于检测Barstar是否插入到基因组目的区域内。
F1：ATTCTCGGCAAAATGCATCAGGACG(SEQ ID NO:12)
R1：TTATGAAGTCTGAAAAGTGAGGAAA(SEQ ID NO:13)
R2：TCTTCATCAACTACCTCCTAT(SEQ ID NO:14)
2.8把筛选到阳性克隆的(F1+R1)PCR产物，切胶回收，用Sanger测序进行验证。测序正确的克隆保存菌种，用于下一步。
3质粒消除
3.1测序正确的阳性克隆在Kan和Spec的双抗平板划线，30℃过夜培养。
3.2挑选单菌落接种至4ml(LB+Kan)的15ml离心管中，并加入4μL 0.5mM IPTG诱导，30℃过夜摇菌。
3.3用上一步菌液划线(LB+Kan)平板，30℃过夜培养。
3.4挑单克隆于(LB+Kan)液体培养基摇菌，30℃培养至浑浊，用菌液分别在(LB+Kan)平板和(LB+Spec)平板上划线，30℃过夜培养，(LB+Spec)平板不长而(LB+Kan)平板正常生长，说明pTarget-sgRNA-donor质粒消除成功。
3.5成功消除pTarget-sgRNA-donor质粒的阳性克隆接种于无抗LB液体培养基37℃摇菌，然后在无抗平板中划线。
3.6在无抗平板上挑单菌落，在无抗LB液体培养基中37℃摇菌，得到的菌液在(LB+Kan)和(LB+Spec)平板上继续划线，30℃培养都不长，说明pTarget-sgRNA-donor质粒和pCas消除都成功了。
3.7用引物(F1+R2)对最后的阳性克隆进行PCR扩增，PCR产物回收后Sanger测序验证，测序正确后保存菌种，用于下一步实验。
实施例2含有barnase基因的质粒转化测试
1.改造后的TOP10F(Barstar)和JM108(Barstar)的大肠杆菌细胞，参照实施例1的2.2到2.4步骤制备转化感受体细胞。
2.含有barnase(序列信息见SEQ ID NO:3)的2个测试质粒(U9041-1和U9041-12，含有Kan抗性基因，全序列见SEQ ID NO:4和5)和对照质粒pUC57-barnase(序列信息见SEQ ID NO:6)，分别化转到TOP10F和JM108，以及改造后的TOP10F(Barstar)和JM108(Barstar)感受体细胞中，在(LB+Kan)的平板上37℃过夜培养。
3.观察转化后的平板长斑数目，并拍照记录，如图5所示，含有barnase的质粒pUC57-barnase，U9041-1和U9041-12转化至改造后的TOP10F(Barstar)和JM108(Barstar)的大肠杆菌细胞中，大肠杆菌均能正常生长产生菌斑；而这3个质粒转化到对照组细胞后，没有产生菌斑(U9041-1)或只有少量菌斑(U9041-12)。具体见下表统计的三种质粒转染改造后大肠杆菌细胞和对照组细胞的平板克隆斑数目。
  TOP10F TOP10F-barstar JM108 JM108-barstar
pUC57-barnase 无克隆斑 >10 3 无克隆斑 >10 3
U9041-1 无克隆斑 >10 3 无克隆斑 ～10 2
U9041-12 <50 >10 3 无克隆斑 >10 3
4.在TOP10F(Barstar)和JM108(Barstar)的平板上随机挑取8个单菌落克隆，过夜摇菌后，用Axygen试剂盒(Corning,AP-MN-P-250)抽提质粒，然后对质粒进行测序验证。测序结果显示barnase基因100％正确。
5.选择测序正确的分别来源于TOP10F Barstar和JM08 Barstar的1个质粒，用HindIII和XbaI做酶切验证，理论上，U9041-1产生3886bp和1644bp条带；U9041-12产生3886bp和801bp条带，其中，3886bp条带是酶切后相同的载体序列条带，644bp和801bp条带分别为包括“Barnase+Halo-tag”和“Barnase”序列的条带，如图6酶切后的电泳图的结果显示酶切完全正确。
序列信息：
SEQ ID NO.1:barstar基因
Figure PCTCN2021099659-appb-000001
SEQ ID NO.2:barstar表达元件序列
Figure PCTCN2021099659-appb-000002
加粗下划线部分代表barstar ORF序列
SEQ ID NO.3:barnase基因
Figure PCTCN2021099659-appb-000003
SEQ ID NO.4:U9041-1质粒全序列
Figure PCTCN2021099659-appb-000004
Figure PCTCN2021099659-appb-000005
Figure PCTCN2021099659-appb-000006
加粗下划线部分代表barnase基因
SEQ ID NO.5:U9041-12质粒全序列
Figure PCTCN2021099659-appb-000007
Figure PCTCN2021099659-appb-000008
加粗下划线部分代表barnase基因
SEQ ID NO.6:pUC57-barnase质粒全序列
Figure PCTCN2021099659-appb-000009
Figure PCTCN2021099659-appb-000010
加粗下划线部分代表barnase基因

Claims (25)

  1. 一种工程菌，其特征在于，所述工程菌的染色体基因组中包含barstar基因，所述工程菌能用于克隆barnase基因。
  2. 根据权利要求1所述的工程菌，其特征在于，所述barstar基因包含SEQ ID NO：1所示核苷酸序列，或与SEQ ID NO：1所示核苷酸序列至少80％一致性的序列。
  3. 根据权利要求1或2所述的工程菌，其特征在于，所述工程菌染色体基因组包括含barstar基因的表达元件。
  4. 根据权利要求3所述的工程菌，所述barstar基因表达元件包括启动子和终止子。
  5. 根据权利要求4所述的工程菌，所述启动子选自EM7、recA、trp、araBAD、TEF1、GAL1、GAL10、lac、psbA、T7或tacP启动子中的一种或多种；所述终止子序列选自MrrnB T1、T7Te、rrnBT1、rrnBT2、rrnB1或rrnB2终止子中的一种或多种。
  6. 根据权利要求3-5中任一项所述的工程菌，所述barstar基因表达元件包含SEQ ID NO:2所示核苷酸序列，或与SEQ ID NO:2所示核苷酸序列至少80％一致性的序列。
  7. 根据权利要求1-6中任一项所述的工程菌，其特征在于，所述工程菌选自大肠杆菌、枯草芽孢杆菌或酵母菌。
  8. 根据权利要求7所述的工程菌，其中，所述大肠杆菌选自TOP10F、JM108、DH5a、stbl3、JM109、DH10b、EPI300或EPI400。
  9. 根据权利要求1-8中任一项所述的工程菌，所述barstar基因克隆到大肠杆菌工程菌染色体中的lacZ，recA，araD，dam，galE，galU，malA，ompT，tonA，rha或者CyaA基因的下游。
  10. 根据权利要求9所述的工程菌，其中，所述barstar基因克隆到大肠杆菌工程菌染色体中CyaA基因的下游。
  11. 一种制备权利要求1-10中任一项所述工程菌的方法，其特征在于，通过基因编辑技术将barstar基因克隆到工程菌的染色体基因组中，所述基因编辑技术选自CRISPR-Cas9技术、锌指核酸酶技术或者转录激活因子样效应物核酸酶技术。
  12. 根据权利要求11中所述的方法，其中，所述基因编辑技术为CRISPR-Cas9技术，包括如下步骤：
    (1)合成左右同源臂序列和barstar基因表达元件序列，将合成的序列插入到含gRNA序列的pTarget质粒，其中，所述左右同源臂序列分别是barstar基因克隆至所述工程菌基因靶序列的左侧和右侧的一段序列；
    (2)步骤(1)得到的质粒与pCas9质粒共转染至工程菌细胞中，培养孵育；
    (3)步骤(2)培养的工程菌通过抗性筛选得到barstar基因整合到染色体基因组中的菌株。
  13. 根据权利要求11或12所述的方法，其中，所述工程菌为大肠杆菌，所述大肠杆菌选自TOP10F、JM108、DH5a、stbl3、JM109、DH10b、EPI300或EPI400。
  14. 根据权利要求12或13所述的方法，所述barstar基因克隆至大肠杆菌工程菌的基因靶序列选自lacZ，recA，araD，dam，galE，galU，malA，ompT，tonA，rha或者CyaA基因的下游序列；优选地克隆至大肠杆菌工程菌基因的靶序列为CyaA基因的下游序列。
  15. 根据权利要求12-14中任一项所述的方法，所述步骤(1)中gRNA是根据barstar基因克隆至工程菌的基因位点设计的。
  16. 根据权利要求12-15中任一项所述的方法，所述步骤(1)中包含gRNA序列的pTarget质粒是以空载体pTarget质粒为模板进行突变、扩增得到的。
  17. 根据权利要求12-16中任一项所述的方法，所述步骤(1)中左右同源臂序列是通过扩增所述工程菌的靶序列左右两侧50-500bp序列得到的。
  18. 根据权利要求12-17中任一项所述的方法，所述步骤(1)中得到的质粒和步骤(2)的pCas9质粒分别包含至少一种抗性基因。
  19. 根据权利要求18所述的方法，所述步骤(2)中包含gRNA的质粒和pCas9质粒的抗性基因分别选自壮观霉素抗性基因、卡那霉素抗性基因、氯霉素抗性基因或氨苄霉素抗性基因。
  20. 根据权利要求12-19中任一项所述的方法，所述步骤(3)中的抗性筛选是指通过双抗性培养基筛选出barstar基因整合到工程菌染色体基因组中的菌株。
  21. 一种克隆barnase基因的方法，其特征在于，所述方法包括将包含barnase基因的质粒转化到权利要求1-10中任一项所述的工程菌或采用权利要求11-20中任一项所述方法制备的工程菌中培养扩增。
  22. 根据权利要求21所述的克隆方法，进一步包括通过双抗性培养基筛选出转化barnase基因的工程菌。
  23. 根据权利要求21或22所述的克隆方法，其特征在于，还包括对扩增培养的工程菌进行质粒提取。
  24. 根据权利要求23所述的克隆方法，进一步包括将提取到的质粒进行序列验证。
  25. 根据权利要求21-24中任一项所述的克隆方法，其特征在于，所述包含barnase基因的质粒选自原核表达载体pET、pGEX系列、酵母载体pRS、pESC系列、植物载体pCAMBIA系列、真核表达载体pcDNA3.1系列、pUC57、U9041-1或者U9041-12。
PCT/CN2021/099659 2020-06-12 2021-06-11 含barstar基因的工程菌及其在barnase基因克隆中的应用 WO2021249536A1 (zh)

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