A method for genome editing in Bacillus subtilis by shortening the length of homology arms

Abstract

The present invention discloses a method for editing the genome of Bacillus subtilis, which belongs to the field of genetic engineering. The present invention uses Bacillus subtilis as an expression host, integrates a single-stranded DNA binding protein and a DNA annealing protein into the Bacillus subtilis genome, and obtains a recombinant Bacillus subtilis, wherein the amino acid sequence of the single-stranded DNA binding protein is shown in SEQ ID NO.1, and the amino acid sequence of the DNA annealing protein is shown in SEQ ID NO.2. By integrating and expressing the single-stranded DNA binding protein and the DNA annealing protein in the Bacillus subtilis genome, the length of the homology arm of the Bacillus subtilis genome integration can be shortened to 35bp, providing a tool for achieving efficient and simple gene editing in Bacillus subtilis.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a Bacillus subtilis genome editing method for shortening the length of homology arms. Background Art

[0002] Bacillus subtilis is a Gram-positive model microorganism with many advantages such as clear genetic background, simple genetic manipulation, easy cultivation, difficult for contaminating phage, no endotoxin and strong secretory protein ability. At the same time, it is approved as a safe (GRAS) food-grade microorganism by the U.S. Food and Drug Administration (FDA). However, compared to the homology arms of 50bp length required for the genetic transformation operation of Escherichia coli, the commonly used homology arms of Bacillus subtilis are 500-1500bp in length, which greatly increases the difficulty of integrating the frame. If the homology arm length is reduced to 50bp, the homology arms can be directly introduced using primers to reduce the difficulty of integrating the frame. However, there is no relevant report at present, therefore, it is urgent to develop an efficient homologous recombination method for shortening the homology arms of Bacillus subtilis. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides a Bacillus subtilis genome editing method that shortens the length of homology arms. This method is achieved by integrating DNA binding proteins and DNA annealing proteins into the genome in advance, inducing them using an IPTG-inducible promoter, and then combining it with a Bacillus subtilis efficient transformation method.

[0004] The first object of the present invention is to provide a method for editing the Bacillus subtilis genome by shortening the length of homology arms, comprising the following steps:

[0005] The single-stranded DNA binding protein and the DNA annealing protein are integrated into the Bacillus subtilis genome. The amino acid sequence of the single-stranded DNA binding protein is shown in SEQ ID NO.1, and the amino acid sequence of the DNA annealing protein is shown in SEQ ID NO.2.

[0006] Specifically, the sequence of SEQ ID NO.1 is:

[0007] MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPF;

[0008] SEQ ID NO.2 sequence is:

[0009] MNQIVKFTDDSGLAVQVTPDDVRRYICENATEKEVGLFLQLCQTQRLNPFVKDAYLVKYGGAPASMITSYQVFNRRACRDANYDGIKSGVVVLRDGDVVHKRGAACYKKAGEELIGGWAEVRFKDGRETAYAEVA LDDYSTGKSNWAKMPGVMIEKCAKAAAWRLAFPDTFQGMYAAEEMDQAQQPEQVRAQAEQPVDLQPIRELFKPYCEHFGITPAEGMTAVCGAVGAEGMHSMTEQQARRARAWMEEEMAAPAVEAEYEVVDEGEVF.

[0010] Furthermore, the single-stranded DNA binding protein and DNA annealing protein are expressed by induction of isopropyl-β-D-thiogalactoside.

[0011] A second object of the present invention is to provide a DNA binding protein and DNA annealing protein expression cassette, wherein the expression cassette comprises a single-stranded DNA binding protein derived from Escherichia coli and a DNA annealing protein derived from an actinomycete phage; wherein the amino acid sequence of the single-stranded DNA binding protein derived from Escherichia coli is shown in SEQ ID NO.1, and the amino acid sequence of the DNA annealing protein derived from an actinomycete phage is shown in SEQ ID NO.2.

[0012] Furthermore, the DNA binding protein and DNA annealing protein expression cassette further comprises an IPTG inducible expression cassette located upstream of the single-stranded DNA binding protein and the DNA annealing protein.

[0013] Furthermore, the IPTG-inducible expression cassette includes an IPTG-inducible promoter and a repressor protein LacI. The expression cassette is induced by isopropyl-β-D-thiogalactopyranoside, that is, the expression induced by isopropyl-β-D-thiogalactopyranoside is regulated by the IPTG-inducible promoter and the IPTG-inducible promoter repressor protein LacI.

[0014] Furthermore, the nucleotide sequence of the IPTG-inducible promoter is shown in SEQ ID NO.3.

[0015] Furthermore, the nucleotide sequence of the repressor protein LacI is shown as SEQ ID NO.4.

[0016] The third object of the present invention is to provide a recombinant Bacillus subtilis with high-efficiency homologous recombination, wherein the recombinant Bacillus subtilis is based on Bacillus subtilis and is integrated with the above-mentioned DNA binding protein and DNA annealing protein expression cassettes.

[0017] Furthermore, the method for constructing the recombinant Bacillus subtilis includes the following steps: integrating ComK protein into Bacillus subtilis, preparing the recombinant strain into competent cells, and integrating the above-mentioned DNA binding protein and DNA annealing protein expression cassettes to obtain the recombinant Bacillus subtilis.

[0018] Furthermore, the nucleotide sequence of the gene encoding the ComK protein is shown in SEQ ID NO. 9. The specific sequence is as follows:

[0019] atgagtcagaaaacagacgcacctttagaatcgtatgaagtgaacggcgcaacaattgccgtgctgccaga

[0020] agaaatagacggcaaaatctgttccaaaattattgaaaaagatgcgtgttttatgtaaacatgaagccgctgcaaatt

[0021] gtcgacagaagctgccgattttttggatcaagctatgcgggaagaaaagcaggaacttatgaagtgacaaaaatttc

[0022] acacaagccgccgatcatggtggacccttcgaaccaaatctttttattccctacactttcttcgacaagaccccaatgc

[0023] ggctggatttcccatgtgcatgtaaaagaattcaaagcgactgaattcgacgatacggaagtgacgttttccaatggg

[0024] aaaacgatggagctgccgatctcttataattcgttcgagaaccaggtataccgaacagcgtggctcagaaccaaatt

[0025] ccaagacagaatcgaccaccgcgtgccgaaaagacaggaatttatgctgtacccgaaagaagagcggacgaaga

[0026] tgatttatgattttattttgcgtgagctcggggaacggtattag

[0027] Furthermore, the ComK protein is expressed by an inducible promoter, which can be any promoter suitable for the host. For example, the present invention uses a xylose-inducible promoter. xylA Regulate expression, the Bacillus subtilis host genome uses a xylose-inducible promoter P xylA Overexpression of an extra copy of the comK gene in the genome.

[0028] Furthermore, the xylose-inducible promoter P xylA The nucleotide sequence is shown in SEQ ID NO. 10. The specific sequence is as follows:

[0029] tttttttaactaaagcttgatctgcaatttgaataataaccactcctttgtttatccaccgaactaagttggtgttttttg

[0030] aagcttgaattagatatttaaaagtatcatatctaatattataactaaattttctaaaaaaaacattgaaataaacatttatttt

[0031] gtatatgatgagataaagttagtttattggataaacaaactaactcaattaagatagttgatggataaacttgttcacttaa

[0032] atcaaagggggaaatgacaaatggtccaaactagtgatatctaaaaatcaaagggggaaatgTTAAAGGA

[0033] GGAAGGATCC.

[0034] Furthermore, the expression cassette sequence of the xylose-inducible promoter expressing the ComK protein is shown in SEQ ID NO.5.

[0035] Furthermore, the steps of preparing competent cells include: activating recombinant Bacillus subtilis that has integrated the ComK protein, inoculating the culture medium in PAB medium for culturing, obtaining a seed solution, inoculating the seed solution in fresh PAB medium for culturing, and adding xylose to induce the formation of Bacillus subtilis competent cells. The Bacillus subtilis DNA transformation method of the present invention is an extremely simple and efficient method, mainly using PAB enriched medium and inducing efficient DNA transformation and gene editing by adding xylose.

[0036] Furthermore, the composition of PAB medium is: peptone 15-25g / L, yeast extract 1-10g / L, beef extract 1-10g / L, NaCl 10-20g / L, KH2PO4 10-20g / L, and K2HPO4 1-10g / L. The inventors have tried LB medium, YN medium, TB medium, DM3G medium, BF medium, and other media, and found that the transformation efficiency of each medium was far lower than that of PAB medium.

[0037] Furthermore, the above transformation method is to add 3% xylose in the early logarithmic growth phase of the strain to induce the competence of the strain.

[0038] Furthermore, the Bacillus subtilis includes but is not limited to Bacillus subtilis WB 600.

[0039] The fourth object of the present invention is to provide applications of the above-mentioned recombinant Bacillus subtilis or DNA binding protein and DNA annealing protein expression cassette in the food and biological fields.

[0040] By means of the above solution, the present invention has at least the following advantages:

[0041] The present invention develops a simple and efficient recombinant Bacillus subtilis transformation method that only requires short homology arms, so that the homology arms are shortened to a minimum of 35bp. The homology arms can be directly introduced using primers, and the construction of an efficient Bacillus subtilis genomic integration library can be achieved.

[0042] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0044] Figure 1The average number of transformants obtained on the plate when the IPTG-inducible promoter was used to express DNA-binding proteins and DNA annealing proteins from different sources, where "-" indicates no induction and "+" indicates induction. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0046] The materials and methods involved in the following examples are as follows:

[0047] 1. The selected DNA binding protein is derived from Escherichia coli (nucleotide sequence is shown in SEQ ID NO.1);

[0048] 2. DNA annealing proteins from different sources include:

[0049] (1) Argonaute protein from Natronobacterium gregoryi (NgAgo, nucleotide sequence shown in SEQ ID NO. 6);

[0050] (2) Beta protein derived from Escherichia coli λ phage (nucleotide sequence shown in SEQ ID NO. 7);

[0051] (3) PapRecT protein from Pseudomonas aeruginosa phage (nucleotide sequence shown in SEQ ID NO. 8);

[0052] (4) CspRecT protein derived from actinomycete phage (Collinsella stercoris phage) (nucleotide sequence shown in SEQ ID NO. 2).

[0053] 3. Seed culture and fermentation of recombinant Bacillus subtilis:

[0054] The Bacillus subtilis was Bacillus subtilis WB600.

[0055] PAB medium composition (g / L): peptone 20, yeast extract 6, beef extract 6, NaCl 14, KH2PO4 14.8, K2HPO4 5.2. After constant volume, adjust the pH to 6.5 and sterilize by autoclaving at 115°C for 20 min.

[0056] Preparation of competent cells: Streak recombinant Bacillus subtilis stored at -80°C onto LB medium plates and incubate at 37°C for 12 hours. Pick a single colony and transfer it to each well of a 24-well plate containing 1 mL of PAB medium. Incubate at 37°C and 220 rpm for 6-8 hours to obtain a seed solution. Inoculate 20% of this seed solution into new PAB medium. Add 50 g / L xylose to the seed solution at a 6% concentration, along with IPTG to a final concentration of 0.5 mM. Incubate at 37°C and 220 rpm for 2 hours to induce the formation of competent cells. Add glycerol to a final concentration of 15% to the competent cells and freeze at -80°C.

[0057] Transformation: Take 100 μL of competent cells and transfer them to a 1.5 mL EP tube to which homologous recombination fragments have been added. Mix well and culture at 37°C and 220 rpm for 1.5 h. Spread the entire tube on the corresponding screening plate.

[0058] Example 1 Homologous recombination cassette

[0059] The construction method is as follows:

[0060] (1) Synthesizing the Kanna resistance gene integration cassette with a 350 bp homology arm sequence shown in SEQ ID NO.9;

[0061] (2) Using it as a template,

[0062] Integration cassette 1 with 35 bp homology arms was amplified using primer 35-F: 5′-cgattgaaacagaagatcacgctgctgctgaataa-3′ and primer 35-R: 5′-gggtttccgcaaacagccaaactgaaagcatatag-3′;

[0063] Integration cassette 2 with 75 bp homology arms was amplified using primer 75-F: 5′-cataatatgacagaagaagaagtaaacgcaagac-3′ and primer 75-R: 5′-gtggtatcaaaatgggaaaaggccttcc-3′;

[0064] Integration cassette 3 with 150 bp homology arms was amplified using primer 150-F: 5′-gaaaatgagtataaatatatgcgcagctgcagc-3′ and primer 150-R: 5′-atcgcgggtgttgaccgcg-3′;

[0065] Integration cassette 4 with 200 bp homology arms was amplified using primer 200-F: 5′-atgtgaactgcgcaaatccatcttgc-3′ and primer 200-R: 5′-ttctggcgactgccaatgctgtagg-3′;

[0066] Integration cassette 5 with 250 bp homology arms was amplified using primer 250-F: 5′-gcatgtgattgtcggcaaggactac-3′ and primer 250-R: 5′-gatgaccagtaaaggcattgcagctg-3′;

[0067] Integration cassette 6 with 350 bp homology arms was amplified using primer 350-F: 5′-gctccagcttatatttatcggtttcttgtatc-3′ and primer 350-R: 5′-tcgtgacatacggaaaagatccagagg-3′.

[0068] Example 2 Construction of recombinant genetically engineered bacteria expressing DNA binding proteins and DNA annealing proteins from different sources

[0069] Genome-integrated xylose-inducible promoter P xylA After the recombinant strain expressing ComK protein (as shown in SEQ ID NO.5) was prepared into competent state, it was transformed with DNA fragments expressing DNA binding protein and DNA annealing protein from different sources, coated on resistant plates and sequenced to obtain the corresponding engineered strain.

[0070] Example 3: Efficient transformation of homology arms after inducing expression of DNA binding proteins and DNA annealing proteins from different sources

[0071] After the recombinant Bacillus subtilis constructed in Example 2 was prepared as described above, the homologous recombination cassette obtained in Example 1 was purified and added to the cells in the same molar ratio for transformation (125 ng of the 35 bp homology arm, 133 ng of the 75 bp homology arm, 148 ng of the 150 bp homology arm, 158 ng of the 200 bp homology arm, 168 ng of the 250 bp homology arm, and 188 ng of the 350 bp homology arm were added). The transformation results are shown in Figure 2. Figure 1As shown (CK group represents Bacillus subtilis strains without the DNA-binding protein and DNA-annealing protein integration cassettes), the results showed that when the DNA-binding protein was selected from Escherichia coli, and the DNA-annealing protein was selected from NgAgo protein from Gregoriana natra, Beta protein from E. coli λ phage, or PapRecT protein from Pseudomonas aeruginosa phage, no transformants were obtained when the homology arm length was shortened to 75 bp and 35 bp, and the number of transformants obtained in all cases was lower than that of the recombinant strain induced to express CspRecT and SSB. However, when IPTG was added to induce the expression of CspRecT and SSB, the maximum number of transformants was obtained for all homology arm lengths, with the maximum increase reaching four times that of the control without additional expression of the recombinant genes. Furthermore, when the homology arm length was shortened to 75 bp and 35 bp, only the recombinant strain induced to express CspRecT and SSB obtained transformants.

[0072] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for editing the genome of Bacillus subtilis by shortening the length of homology arms, characterized in that: The following steps are involved: The single-stranded DNA binding protein and the DNA annealing protein are integrated into the Bacillus subtilis genome. The amino acid sequence of the single-stranded DNA binding protein is shown in SEQ ID NO.1, and the amino acid sequence of the DNA annealing protein is shown in SEQ ID NO.

2.

2. The method for editing the Bacillus subtilis genome according to claim 1, wherein: The single-stranded DNA binding protein and the DNA annealing protein are expressed by induction of isopropyl-β-D-thiogalactoside.

3. A DNA binding protein and DNA annealing protein expression cassette, characterized in that: It includes a gene sequence encoding a single-stranded DNA binding protein derived from Escherichia coli and a gene sequence encoding a DNA annealing protein derived from an actinomycete phage; wherein the amino acid sequence of the single-stranded DNA binding protein derived from Escherichia coli is shown as SEQ ID NO.1, and the amino acid sequence of the DNA annealing protein derived from an actinomycete phage is shown as SEQ ID NO.

2.

4. The DNA binding protein and DNA annealing protein expression cassette according to claim 3, characterized in that: The DNA binding protein and DNA annealing protein expression cassette also includes an IPTG-induced expression frame located upstream of the single-stranded DNA binding protein encoding gene sequence and the DNA annealing protein encoding gene sequence. The IPTG-induced expression frame includes an IPTG-inducible promoter and a repressor protein LacI encoding gene sequence.

5. The DNA binding protein and DNA annealing protein expression cassette according to claim 4, characterized in that: The nucleotide sequence of the IPTG-inducible promoter is shown in SEQ ID NO.3; the nucleotide sequence of the repressor protein LacI encoding gene sequence is shown in SEQ ID NO.

4.

6. A recombinant Bacillus subtilis with high efficiency homologous recombination, characterized by: The recombinant Bacillus subtilis is integrated with the DNA binding protein and DNA annealing protein expression cassette according to any one of claims 3 to 5.

7. The recombinant Bacillus subtilis according to claim 6, characterized in that The method for constructing the recombinant Bacillus subtilis comprises the following steps: integrating ComK protein into Bacillus subtilis, preparing the recombinant strain into competent cells, and then integrating the DNA binding protein and DNA annealing protein expression cassettes to obtain the recombinant Bacillus subtilis.

8. The recombinant Bacillus subtilis according to claim 7, characterized in that: The steps of preparing competent cells include: activating the recombinant Bacillus subtilis integrated with ComK protein, inoculating it into PAB medium for cultivation to obtain seed liquid, inoculating the seed liquid into new PAB medium for cultivation, and inducing the formation of Bacillus subtilis competent cells.

9. The recombinant Bacillus subtilis according to claim 8, characterized in that: The PAB culture medium is composed of: 15-25 g / L peptone, 1-10 g / L yeast powder, 1-10 g / L beef extract, 10-20 g / L NaCl, 10-20 g / L KH2PO4, and 1-10 g / L K2HPO4.

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