CN114990035A - Bacillus subtilis BS168 competent engineering strain and application thereof - Google Patents
Bacillus subtilis BS168 competent engineering strain and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis BS168 competence engineering strain and application thereof, wherein the bacillus subtilis BS168-P xylA -comK recombinant strain with P inserted in Amye site xylA comK gene and does not contain a resistance tag. Bacillus subtilis BS168-P in the invention xylA The comK recombinant strain has stronger plasmid transformation effect compared with a BS168 wild strain, can be compatible with various different site recombinations, and can be used as an original strain of an excellent engineering strain due to low modification degree. Moreover, the strain has low modification cost and extremely high application prospect.
Description
Technical Field
The invention belongs to the field of construction of genetic engineering strains, and particularly relates to a bacillus subtilis BS168 competence engineering strain and application thereof.
Background
The bacillus subtilis belongs to gram-positive aerobic bacteria, and has wide application in the aspects of agriculture, medical treatment, food biology and other technologies and recombinant protein production. However, in the related art, although the Bacillus subtilis 168(Bacillus subtilis 168) strain has the capability of forming natural competence, the transformation rate and success rate of competent cells are still low, which greatly limits the application of the strain in protein engineering and virtually increases the difficulty of metabolic engineering. And another related art Bacillus subtilis SCK6(ErmR, 1A751 derived, lacA:: P) with super transformation ability xylA comK) strain, although the transformation success rate is improved to a certain extent compared with that of the bacillus subtilis 168, the grinding of neomycin resistance genes contained in the comK strain greatly limits the application prospect of the comK strain as probiotics in the aspect of live bacteria. Meanwhile, compared with the bacillus subtilis 168, the SCK6 is a multigenic modified (his nprR2 nprE18 delta DaprA3 delta DeglS102 delta DbgltbglSRV) strain, so that the SCK6 is not suitable to be used as an original strain of an excellent engineering strain, and causes great limitation on the practical application of the strain.
Therefore, the development of the bacillus subtilis competent engineering strain which can effectively overcome the defects of the strain and has wider applicability and stronger transformation success rate is of great significance for the deep utilization of the bacillus subtilis.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the bacillus subtilis BS168 competence engineering strain and the application thereof are provided, the engineering strain does not contain a resistance label, is obtained by conventional bacillus subtilis 168 transformation, does not have polygene transformation, can be used as an original strain of an excellent engineering strain, has higher plasmid transformation and gene recombination efficiency compared with the conventional bacillus subtilis 168, and can efficiently transform various plasmids.
In the first aspect of the invention, the invention provides a bacillus subtilis BS168-P xylA comK recombinant strain, Bacillus subtilis BS168-P xylA -comK recombinant strain with P inserted in Amye site xylA -comK gene.
In some embodiments of the invention, the Bacillus subtilis BS168-P xylA The comK recombinant strain does not contain a resistance tag.
In some embodiments of the invention, the Bacillus subtilis BS168-P xylA The nucleotide sequence of the comK recombinant strain is shown as SEQ ID NO: shown at 20.
In the invention, the Bacillus subtilis BS168-P xylA The comK recombinant strain does not contain any resistance tag and has few inserted modified genes, so that the comK recombinant strain can be used as an original strain of an excellent engineering strain. Furthermore, the inventor verifies through experiments that the bacillus subtilis BS168-P xylA Compared with the original bacillus subtilis strain, the comK recombinant strain has stronger plasmid transformation and gene recombination efficiency, can easily realize various gene site editing or transformation such as Amye site recombination, Upp site recombination and the like, and has extremely high application value.
In a second aspect of the invention, there is provided Bacillus subtilis BS168-P according to the first aspect of the invention xylA -a method for constructing a comK recombinant strain, comprising the steps of:
(1) construction of pDG1730-P grac -Phesm recombinant plasmid, transformation of Bacillus subtilisBacillus BS168 to obtain bacillus subtilis BS 168-Spc-Phesm;
(2) construction of resistance tag-free pDG1730 (. DELTA.Spc) -P xylA -comK recombinant plasmid, transforming Bacillus subtilis BS 168-Spc-Phesm;
(3) screening the positive strain with the 4CP resistance, namely the bacillus subtilis BS168-P xylA -comK recombinant strain.
In some embodiments of the invention, the pDG1730-P grac -the nucleotide sequence of the Phesm gene in the Phesm recombinant plasmid is shown in SEQ ID NO: shown at 7.
In the invention, the Phesm gene is obtained by codon optimization. Wherein, the amplification primers for amplifying the Phesm gene are as follows:
upstream Phesm-F: 5'-CCCATATAAAGGAGGAAGGATCCATGGAAGAAAAGTTAAAGCAAC-3' and downstream Phesm-R: 5'-ACGGATATCATCATCGCTCATATTAAGCTTGCTTGAACTGACT-3' are provided.
In some embodiments of the invention, the resistance tags include an ampicillin resistance tag, a spectinomycin resistance tag, and a chloramphenicol resistance tag.
In some preferred embodiments of the invention, the resistance tag is a spectinomycin resistance tag.
In some embodiments of the invention, the pDG1730-P grac -Phesm recombinant plasmid was prepared by linearizing fragment pDG1730, P grac The amplified fragment and the Phesm amplified fragment are obtained by multi-fragment connection and cyclization. Wherein, the ligation and circularization of the nucleic acid fragments can be achieved by a kit or conventional methods conventional in the art, including but not limited to a multi-fragment seamless cloning kit.
In some preferred embodiments of the invention, the pDG1730 linearized fragment is amplified using the pDG1730 plasmid as a template, wherein the amplification primers are: upstream PDG 1730-F: 5'-ATGAGCGATGATGATATCCGT-3' and downstream PDG 1730-R: 5'-GTCGAGATCCCCCTATGCAA-3' are provided.
In some preferred embodiments of the invention, P grac The amplified fragment is obtained by amplifying pHT254 plasmid as a template, wherein the amplification primers are as follows:upstream Pgrac-F: 5'-TTGCATAGGGGGATCTCGACCGGAAGGAAATGATGACCTC-3' and downstream Pgrac-R: 5'-GGATCCTTCCTCCTTTATATGGG-3' are provided.
In some embodiments of the invention, the resistance tag-free pDG1730(Δ Spc) -P xylA The comK recombinant plasmid was obtained by linearizing fragment pDG1730 (. DELTA.Spc), P xylA And the amplified fragment and the comK amplified fragment are obtained by multi-fragment connection and cyclization. Wherein, the ligation and circularization of the nucleic acid fragments can be achieved by a kit or conventional methods conventional in the art, including but not limited to a multi-fragment seamless cloning kit.
In some preferred embodiments of the invention, the pDG1730(Δ Spc) linearized fragment is amplified using the pDG1730 plasmid as a template, wherein the amplification primers are: upstream pDG1730(Δ Spc) -F: 5'-ATGAGCGATGATGATATCCGTT-3' and downstream pDG1730(Δ Spc) -R: 5'-TCACGAACGAAAATCGCCAT-3' are provided.
In some preferred embodiments of the invention, P xylA The amplified fragment is obtained by amplifying the Bacillus subtilis BS168 genome DNA serving as a template, wherein the amplification primers are as follows: upstream PxylA-F: 5 'AATGGCGATTTTCGTTCGTGAGCGATATCCACTTCATCCACT-3' and downstream PxylA-R: 5 'CATATTATGGCCTCCTTAAAAATAAATTCATTCAAATAC-3'.
In some preferred embodiments of the present invention, the comK amplified fragment is amplified using bacillus subtilis BS168 genomic DNA as a template, wherein the amplification primers are: upstream comK-F: 5'-TTTTTAAGGAGGCCATAATATGAGTCAGAAAACAGACGCACC-3' and downstream comK-R: 5'-AACGGATATCATCATCGCTCATCTAATACCGTTCCCCGAGC-3' are provided.
In a third aspect of the invention, there is provided a cell product comprising Bacillus subtilis BS168-P according to the first aspect of the invention xylA -comK recombinant strain.
In some embodiments of the invention, the cell product comprises a cell (or bacteria) suspension, lyophilized, or other conventional form.
In a fourth aspect of the invention, there is provided the use of a cell product according to the third aspect of the invention in the preparation of a food, pharmaceutical or cosmetic product.
In a fifth aspect of the present invention, there is provided Bacillus subtilis BS168-P according to the first aspect of the present invention xylA -application of comK recombinant strain in construction of Bacillus subtilis recombinant strain.
In the invention, the inventor verifies the Bacillus subtilis BS168-P through experiments xylA The comK recombinant strain can be used as an original strain of an excellent engineered strain, and is therefore based on the Bacillus subtilis BS168-P xylA It is entirely predictable that the comK recombinant strain is a template for gene modification to obtain a Bacillus subtilis recombinant strain with similar effect.
In a sixth aspect of the present invention, there is provided Bacillus subtilis BS168-P according to the first aspect of the present invention xylA Application of comK recombinant strain in construction of plasmid transformation vector and gene modification.
In the invention, the inventor verifies the Bacillus subtilis BS168-P through experiments xylA The comK recombinant strain can be used as an original strain of an excellent engineering strain and has better plasmid transformation and gene recombination functions, so that the Bacillus subtilis BS168-P is used xylA The comK recombinant strain is completely expected as a target strain for transforming various types of plasmids with a plasmid vector to obtain a high transformation rate.
The invention has the beneficial effects that:
1. p is inserted into AmyE locus of Bacillus subtilis BS168 genome xylA The comK gene box artificially controls the BS168 wild strain to become competent cells under the condition of not introducing resistance genes, and maintains the competent state through ultralow temperature preservation, thereby greatly simplifying the modification process of the wild strain BS168 and reducing the modification cost.
2. Bacillus subtilis BS168-P in the invention xylA Compared with a BS168 wild strain, the comK recombinant strain has stronger plasmid transformation effect and fragment recombination capability, can be compatible with various different site recombinations, and can be used as an original strain of an excellent engineering strain due to low modification degree.
Drawings
FIG. 1 is pDG1730-P grac Schematic construction scheme of the Phesm recombinant plasmid.
FIG. 2 is a diagram of the growth of the BS168-Spc-Phesm recombinant strain and wild strain in LB-4CP solid medium.
FIG. 3 is a gel electrophoresis of the amplification product of the BS168-Spc-Phesm recombinant strain, and the band A is the amplification product.
FIG. 4 is pDG1730 (. DELTA.Spc) -P xylA Schematic construction scheme of comK recombinant plasmid.
FIG. 5 is pDG1730 (. DELTA.Spc) -P xylA Gel electrophoresis of the amplification product of the comK recombinant plasmid, and the band B is the amplification product.
FIG. 6 shows BS168-P xylA Gel electrophoresis of amplification product of comK recombinant strain, band C being amplification product of non-recombinant Bacillus subtilis BS168 standard strain, band D being BS168-P xylA Amplification products of comK recombinant strains.
FIG. 7 is a BS168-P xylA Schematic representation of the construction principle of comK recombinant strains.
FIG. 8 shows Bacillus subtilis BS168-P xylA The result of the recombination function verification of comK recombinant strains at the Amye site, wherein a is the growth condition of the non-recombined bacillus subtilis BS168 standard strain on an Spc resistant plate, and b is bacillus subtilis BS168-P xylA Growth of comK recombinant strains on Spc-resistant plates, c being BS168-P xylA Gel electrophoresis of amplification products of comK recombinant strains using amyE 2-F and amyE 2-R.
FIG. 9 shows Bacillus subtilis BS168-P xylA The result of functional verification of recombination of comK recombinant strain at Upp site, wherein a is growth of non-recombined Bacillus subtilis BS168 standard strain on cm resistant plate, and b is Bacillus subtilis BS168-P xylA Growth of comK recombinant strains on cm-resistant plates, c being BS168-P xylA Gel electrophoresis of amplification products of the comK recombinant strain using Upp1-F and Upp 2-R.
FIG. 10 shows Bacillus subtilis BS168-P xylA -plasmid transformation functional verification of comK recombinant strain, wherein a is unreinforcedGroup of Bacillus subtilis BS168 standard strain and blank pHT254 transformation BS168-P xylA -comparison of growth of comK recombinant strains on cm resistant plates; b is the non-recombinant Bacillus subtilis BS168 standard strain and blank pHT43 transformed BS168-P xylA Growth of comK recombinant strains on cm-resistant plates.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Test materials
In the following examples, the composition of the 2 XYT liquid medium used was: tryptone 16g \ L, yeast extract 10g \ L, sodium chloride 5g \ L. The preparation method comprises the following steps: mixing the above components, and sterilizing at 121 deg.C for 20 min.
The composition of the LB solid medium used was: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium chloride and 15 g/L agar powder. The preparation method comprises the following steps: mixing the above components, and sterilizing at 121 deg.C for 20 min.
The LB-4CP solid medium used had the composition: 10 g/L of tryptone, 5 g/L of yeast extract, 5 g/L of sodium chloride, 4 g/L of 4-chlorophenylalanine and 15 g/L of agar powder. The preparation method comprises the following steps: mixing the above components, and sterilizing at 121 deg.C for 20 min.
The composition of the electroporation buffer used was: 0.5M trehalose, 0.5M sorbitol, 0.5M mannitol, 0.5mM MgCl 2 ，0.5mM K 2 HPO 4 ，0.5mM KH 2 PO 4 。
The plasmid extraction kit, the bacterial DNA extraction kit and the glue recovery kit are all purchased from Guangzhou Meiji Biotechnology GmbH; the PCR amplification enzyme premix kit is purchased from Nanjing Nuozantin Biotechnology Co., Ltd; the seamless cloning kit is purchased from Shanghai Biotechnology engineering, Inc.
Bacillus subtilis BS168-P xylA Preparation of recombinant strain of comK
The bacillus subtilis BS168 competent engineering strain in the embodiment of the invention is a bacillus subtilis BS168 competent strain without antibiotic label and with super-natural transformation capability (named as BS 168-P) xylA -comK). The engineering strain is prepared by introducing bacillus subtilis competence formation key transcription factor comK to an Amye site in a genetic engineering way in a traceless manner, can realize efficient, rapid and low-cost plasmid transformation and gene editing, and has no resistance label.
(1) Construction of pDG1730-P grac -Phesm recombinant plasmid:
according to P published in existing databases grac And a Phesm gene sequence, designing primers with corresponding homologous sequences, and respectively amplifying. Meanwhile, primers were designed for pDG1730 plasmid (Bacillus subtilis integration vector pDG1730, purchased from Wuhan vast Ling Biotech Co., Ltd.), and pDG1730 linearized fragments were amplified.
The specific primer sequences are as follows:
in this example, the pDG1730 linearized fragment was amplified using pDG1730 plasmid as template, wherein the amplification primers were:
upstream PDG 1730-F: 5'-ATGAGCGATGATGATATCCGT-3' (SEQ ID NO: 1);
downstream PDG 1730-R: 5'-GTCGAGATCCCCCTATGCAA-3' (SEQ ID NO: 2).
In this embodiment, P grac The amplified fragment is obtained by using pHT254 plasmid (purchased from Wuhan vast Ling Biotech limited) as a template, wherein the amplification primers are as follows:
upstream Pgrac-F: 5'-TTGCATAGGGGGATCTCGACCGGAAGGAAATGATGACCTC-3' (SEQ ID NO: 3);
downstream Pgrac-R: 5'-GGATCCTTCCTCCTTTATATGGG-3' (SEQ ID NO: 4).
In this example, the Phesm amplified fragment is obtained by amplifying a codon-optimized Phesm gene sequence as a template, wherein the amplification primers are:
upstream Phesm-F: 5'-CCCATATAAAGGAGGAAGGATCCATGGAAGAAAAGTTAAAGCAAC-3' (SEQ ID NO: 5);
downstream Phesm-R: 5'-ACGGATATCATCATCGCTCATATTAAGCTTGCTTGAACTGACT-3' (SEQ ID NO: 6).
The nucleotide sequence of the amplified Phesm fragment is as follows: 5'-ATGGAAGAAAAGTTAAAGCAACTTGAACAAGAAGCGCTTGAGCAAGTCGAAGCCGCCAGCAGTTTGAAAGTTGTGAACGACATCAGAGTCCAGTATCTTGGTAAGAAGGGCCCTATCACTGAAGTCCTGCGGGGAATGGGCAAATTAAGCGCAGAGGAAAGACCTAAGATGGGCGCCTTGGCAAACGAAGTACGTGAGAGAATAGCAAATGCGATAGCAGATAAAAACGAGAAGTTGGAGGAGGAGGAGATGAAACAAAAACTGGCAGGTCAGACAATAGATGTAACCCTTCCGGGCAACCCTGTGGCAGTTGGGGGACGCCATCCGCTCACGGTGGTCATTGAAGAGATAGAAGACTTATTCATTGGTATGGGATATACAGTTGAAGAAGGACCTGAAGTAGAAACAGATTACTATAACTTCGAGAGTCTTAATTTACCAAAAGAGCATCCAGCACGCGACATGCAAGACTCCTTCTACATTACGGAAGAGACCCTCATGCGTACACAGACAAGTCCAGTTCAGACTCGCACAATGGAGAAACACGAAGGCAAAGGCCCGGTGAAAATAATTTGTCCAGGAAAAGTTTACCGGCGGGACAATGATGATGCAACTCATTCACATCAGTTTATGCAGATCGAGGGATTGGTTGTTGATAAAAACATCTCAATGAGTGACCTCAAGGGTACCTTAGAATTAGTGGCTAAAAAAATGTTTGGACAGGACAGAGAGATCCGCCTGAGACCATCCTTCTTTCCTTTTACTGAGCCGTCAGTTGAAGTTGATGTGACTTGCTTCAAATGCGGCGGCAACGGCTGCTCAGTCTGTAAAGGCACCGGATGGATAGAGATTTTGGGGGCCGGCATGGTACATCCGAACGTGCTGAAAATGGCAGGGTTCGATCCGAAAGAGTATCAGGGATTTGGCTTTGGAATGGGAGTGGAGCGCATAGCTATGCTGAAGTATGGAATTGATGATATTCGCCATTTCTATACGAATGACGTCCGCTTCATTAGTCAGTTCAAGCAAGCTTAA-3' (SEQ ID NO: 7).
The pDG1730 linearized fragment obtained in the above step, P, was cloned using a multi-fragment seamless cloning kit grac The amplified fragment and the Phesm amplified fragment are subjected to multi-fragment connection and cyclization to obtain pDG1730-P grac -Phesm recombinant plasmid, scheme as shown in FIG. 1.
(2)pDG1730-P grac -transformation, extraction and identification of the Phesm recombinant plasmid:
the pDG1730-P obtained in the step (1) grac Transforming the recombinant plasmid of Phesm into competent cells of Escherichia coli DH5 alpha, and selecting positive transformants from the competent cells for amplification cultureExtracting plasmid, sequencing and identifying, and storing the correctly identified recombinant plasmid at-20 deg.C for use.
(3)pDG1730-P grac -transformation of the bacillus subtilis BS168 strain with the Phesm recombinant plasmid:
the pDG1730-P obtained in the step (2) grac The recombinant plasmid of Phesm is transformed into a Bacillus subtilis BS168 strain (purchased from Beiner Biotechnology Co., Ltd.) by electric shock, a positive strain with resistance to spectinomycin (Spc) is screened, the functionality of the Phesm gene in the positive strain is determined by using 4-chlorophenylalanine (4CP), and glycerol conservation at-80 ℃ is carried out on the positive strain (BS168-Spc-Phesm) which is verified to be correct by sequencing and has normal functions of the Phesm gene.
The method comprises the following specific steps:
bacillus subtilis BS168 was cultured overnight in 2 XYT medium. The overnight cultured bacterial suspension was diluted 100-fold with fresh 2 XYT medium and cultured to OD600 of 0.8. Adding 1% DL-threonine, 2% glycine, 0.1% tryptophan and 0.03% Tween 80 by mass, shaking for 2 hr, and cooling on ice for 15 min. Centrifuge at 4000 Xg for 10 min. Discard the supernatant, resuspend it in electroporation buffer, centrifuge at 6000 Xg for 10min, repeat 3 times. Resuspend with a small volume of electroporation buffer. Pipette 100. mu.L of resuspension, add 2. mu.L of pDG1730-P grac Phesm recombinant plasmid (100 ng/. mu.L), for shock transformation (shock conditions 1250V/mm, 200. omega., 25. mu.F). Immediately after the completion of transformation, 1mL of 2 XYT medium supplemented with 0.5M sorbitol and 0.38M mannitol was added, shaken at 37 ℃ and 200rpm for 3 hours, and spread on an Spc-resistant selection LB solid medium for selection. The screening situation is shown in FIG. 2.
It can be found that the positive BS168-Spc-Phesm recombinant strain can not grow on LB-4CP solid medium, while the non-recombinant Bacillus subtilis BS168 standard strain can grow.
The verification of the positive BS168-Spc-Phesm recombinant strain is realized by adopting PCR amplification, and the used primers are as follows:
upstream amyE 1-F: 5'-GGAAGGGAATCAAGGAGATAAAAG-3' (SEQ ID NO: 8);
downstream amyE 1-R: 5'-CAGGATAAAGCACAGCTACAGAC-3' (SEQ ID NO: 9).
The PCR amplification products were checked for band size using gel electrophoresis, and the results are shown in FIG. 3. In FIG. 3, band A is the amplification product of the above primer, and the fragment size is 3100 bp.
(4) Construction of pDG1730 (. DELTA.Spc) -P xylA -comK recombinant plasmid:
designing primers with corresponding homologous sequences, and respectively amplifying to obtain P xylA Amplified fragments, comK amplified fragments, and pDG1730 linearized fragments (pDG1730(Δ Spc)) without spectinomycin (Spc) resistance tag.
The specific primer sequences are as follows:
in this example, the pDG1730(Δ Spc) linearized fragment was amplified using pDG1730 plasmid as a template, wherein the amplification primers were:
upstream pDG1730(Δ Spc) -F: 5'-ATGAGCGATGATGATATCCGTT-3' (SEQ ID NO: 10);
downstream pDG1730(Δ Spc) -R: 5'-TCACGAACGAAAATCGCCAT-3' (SEQ ID NO: 11).
In this embodiment, P xylA The amplified fragment is obtained by amplifying bacillus subtilis BS168 genome DNA serving as a template (extracted from the bacillus subtilis BS168 strain), wherein amplification primers are as follows:
upstream PxylA-F: 5'-AATGGCGATTTTCGTTCGTGAGCGATATCCACTTCATCCACT-3' (SEQ ID NO: 12);
downstream PxylA-R: 5'-CATATTATGGCCTCCTTAAAAATAAATTCATTCAAATAC-3' (SEQ ID NO: 13).
In this example, the comK amplified fragment was amplified using Bacillus subtilis BS168 genomic DNA as a template (extracted from Bacillus subtilis BS168 strain described above), wherein the amplification primers were:
upstream comK-F: 5'-TTTTTAAGGAGGCCATAATATGAGTCAGAAAACAGACGCACC-3' (SEQ ID NO: 14);
downstream comK-R: 5'-AACGGATATCATCATCGCTCATCTAATACCGTTCCCCGAGC-3' (SEQ ID NO: 15).
The pDG1730 (. DELTA.Spc) linearized fragment obtained in the above procedure, P, using a multiple fragment seamless cloning kit xylA Amplified fragment and comK amplified fragmentMulti-fragment ligation and cyclization to give pDG1730 (. DELTA.Spc) -P xylA The comK recombinant plasmid, the scheme of which is shown in FIG. 4.
(5)pDG1730(ΔSpc)-P xylA -transformation, extraction and identification of comK recombinant plasmids:
the pDG1730 (. DELTA.Spc) -P obtained in the step (4) xylA The comK recombinant plasmid is transformed into escherichia coli DH5 alpha competent cells, positive transformants are selected from the comK recombinant plasmid for amplification culture, the plasmid is extracted, sequencing identification is carried out, and the correctly identified recombinant plasmid is stored at the temperature of minus 20 ℃ for later use.
Wherein, FIG. 5 shows pDG1730 (. DELTA.Spc) -P xylA Gel electrophoresis of PCR-verified product of a part of DNA sequence in comK recombinant plasmid, band B is recombinant plasmid pDG1730 (. DELTA.Spc) -P xylA comK verification band with fragment size 946 bp.
The amplification primers are as follows:
upstream PxylA-comK-F: 5'-CATTGAATGACGGGGCAGAC-3' (SEQ ID NO: 16).
Upstream PxylA-comK-R: 5'-GACTCTTCATCATCATTGGC-3' (SEQ ID NO: 17).
(6)pDG1730(ΔSpc)-P xylA comK recombinant plasmid transformation of BS168 engineered strain:
the pDG1730 (delta Spc) -P obtained in the step (5) xylA The comK recombinant plasmid is transformed into a BS168-Spc-Phesm recombinant strain (the positive strain obtained in the step (3)) through electric shock, a positive strain with 4CP resistance is screened (the Phesm gene is generated, if the Phesm gene is replaced by a repair template, the Phesm gene can grow on a 4CP culture medium), and a correct strain with positive 4CP resistance is verified through sequencing, namely the Bacillus subtilis BS168-P xylA -comK recombinant strain. Preserving the seeds with glycerol at the temperature of minus 80 ℃.
The specific operation steps are the same as the step (3), wherein in the embodiment, the verification primer is:
upstream amyE 2-F: 5'-GTTGACGCGGTCATCAATC-3' (SEQ ID NO: 18);
downstream amyE 2-R: 5'-TGTCCAGCCATCACATTGTG-3' (SEQ ID NO: 19).
The PCR amplification products were checked for band size using gel electrophoresis, and the results are shown in FIG. 6. Drawing (A)In 6, the band C is an amplification product of the non-recombinant bacillus subtilis BS168 standard strain based on the primers, and the size is 750 bp; strip D is BS168-P xylA The comK recombinant strain is based on the amplification product of the above primers and has a size of 1364 bp.
Wherein the obtained Bacillus subtilis BS168-P xylA -the gene sequence of the comK recombinant strain is: 5' -ATGTTTGCAAAACGATTCAAAACCTCTTTACTGCCGTTATTCGCTGGATTTTTATTGCTGTTTCATTTGGTTCTGGCAGGACCGGCGGCTGCGAGTGCTGAAACGGCGAACAAATCGAATGAGCTTACAGCACCGTCGATCAAAAGCGGAACCATTCTTCATGCATGGAATTGGTCGTTCAATACGTTAAAACACAATATGAAGGATATTCATGATGCAGGATATACAGCCATTCAGACATCTCCGATTAACCAAGTAAAGGAAGGGAATCAAGGAGATAAAAGCATGTCGAACTGGTACTGGCTGTATCAGCCGACATCGTATCAAATTGGCAACCGTTACTTAGGTACTGAACAAGAATTTAAAGAAATGTGTGCAGCCGCTGAAGAATATGGCATAAAGGTCATTGTTGACGCGGTCATCAATCATACCACCAGTGATTATGCCGCGATTTCCAATGAGGTTAAGAGTATTCCAAACTGGACACATGGAAACACACAAATTAAAAACTGGTCTGATCGATGGGATGTCACGCAGAATTCATTGCTCGGGCTGTATGACTGGAATACACAAAATACACAAGTACAGTCCTATCTGAAACGGTTCTTAGACAGGGCATTGAATGACGGGGCAGACGGTTTTCGATTTGATGCCGCCAAACATATAGAGGCGATATCCACTTCATCCACTCCATTTGTTTAATCTTTAAATTAAGTATCAACATAGTACATAGCGAATCTTCCCTTTATTATATCTAATGTGTTCATAAAAAACTAAAAAAAATATTGAAAATACTGACGAGGTTATATAAGATGAAAATAAGTTAGTTTGTTTAAACAACAAACTAATAGGTGATGTACTTACTATATGAAATAAAATGCATCTGTATTTGAATGAATTTATTTTTAAGGAGGCCATAATATGAGTCAGAAAACAGACGCACCTTTAGAATCGTATGAAGTGAACGGCGCAACAATTGCCGTGCTGCCAGAAGAAATAGACGGCAAAATCTGTTCCAAAATTATTGAAAAAGATTGCGTGTTTTATGTAAACATGAAGCCGCTGCAAATTGTCGACAGAAGCTGCCGATTTTTTGGATCAAGCTATGCGGGAAGAAAAGCAGGAACTTATGAAGTGACAAAAATTTCACACAAGCCGCCGATCATGGTGGACCCTTCGAACCAAATCTTTTTATTCCCTACACTTTCTTCGACAAGACCCCAATGCGGCTGGATTTCCCATGTGCATGTAAAAGAATTCAAAGCGACTGAATTCGACGATACGGAAGTGACGTTTTCCAATGGGAAAACGATGGAGCTGCCGATCTCTTATAATTCGTTCGAGAACCAGGTATACCGAACAGCGTGGCTCAGAACCAAATTCCAAGACAGAATCGACCACCGCGTGCCGAAAAGACAGGAATTTATGCTGTACCCGAAAGAAGAGCGGACGAAGATGATTTATGATTTTATTTTGCGTGAGCTCGGGGAACGGTATTAGACGGACAAGCTAGTGACATGGGTAGAGTCGCATGATACGTATGCCAATGATGATGAAGAGTCGACATGGATGAGCGATGATGATATCCGTTTAGGCTGGGCGGTGATAGCTTCTCGTTCAGGCAGTACGCCTCTTTTCTTTTCCAGACCTGAGGGAGGCGGAAATGGTGTGAGGTTCCCGGGGAAAAGCCAAATAGGCGATCGCGGGAGTGCTTTATTTGAAGATCAGGCTATCACTGCGGTCAATAGATTTCACAATGTGATGGCTGGACAGCCTGAGGAACTCTCGAACCCGAATGGAAACAACCAGATATTTATGAATCAGCGCGGCTCACATGGCGTTGTGCTGGCAAATGCAGGTTCATCCTCTGTCTCTATCAATACGGCAACAAAATTGCCTGATGGCAGGTATGACAATAAAGCTGGAGCGGGTTCATTTCAAGTGAACGATGGTAAACTGACAGGCACGATCAATGCCAGGTCTGTAGCTGTGCTTTATCCTGATGATATTGCAAAAGCGCCTCATGTTTTCCTTGAGAATTACAAAACAGGTGTAACACATTCTTTCAATGATCAACTGACGATTACCTTGCGTGCAGATGCGAATACAACAAAAGCCGTTTATCAAATCAATAATGGACCAGAGACGGCGTTTAAGGATGGAGATCAATTCACAATCGGAAAAGGAGATCCATTTGGCAAAACATACACCATCATGTTAAAAGGAACGAACAGTGATGGTGTAACGAGGACCGAGAAATACAGTTTTGTTAAAAGAGATCCAGCGTCGGCCAAAACCATCGGCTATCAAAATCCGAATCATTGGAGCCAGGTAAATGCTTATATCTATAAACATGATGGGAGCCGAGTAATTGAATTGACCGGATCTTGGCCTGGAAAACCAATGACTAAAAATGCAGACGGAATTTACACGCTGACGCTGCCTGCGGACACGGATACAACCAACGCAAAAGTGATTTTTAATAATGGCAGCGCCCAAGTGCCCGGTCAGAATCAGCCTGGCTTTGATTACGTGCTAAATGGTTTATATAATGACTCGGGCTTAAGCGGTTCTCTTCCCCATTGA-3’(SEQ ID NO：20)。
The schematic diagram of the recombinant strain construction principle in the above steps is shown in FIG. 7.
Bacillus subtilis BS168-P xylA Verification of the recombination efficiency of comK recombinant strains
(1) Verification of Amye site recombination function:
taking the above BS168-P xylA The comK recombinant strain was inoculated in 2 XYT medium and cultured overnight at 37 ℃ at 220 r/min. The overnight culture of bacterial liquid with fresh 2 xYT medium for 100 times dilution, 37 degrees C, 220r/min shake bacteria culture to OD600 of 2.0. Xylose was added at 1.5% by mass, and the suspension was diluted with fresh 2 xyt medium to OD600 ═ 1.0. Culturing at 37 deg.C and 220r/min for 2 hr to obtain competent cell solution. mu.L of pDG1730 plasmid with the concentration of 200 ng/mu.L is taken and added into 100 mu.L of competent cell solution, the mixture is uniformly mixed, incubated for 90min at 37 ℃ and 220r/min, coated on an Spc (140 mu g/mL) resistant plate, inverted overnight culture is carried out at 37 ℃, and positive colonies are screened. As a control, a standard strain of non-recombinant Bacillus subtilis BS168 was used.
Meanwhile, PCR amplification verification was performed using amyE 2-F and amyE 2-R.
The results are shown in FIG. 8.
It can be found that BS168-P xylA The homologous recombination efficiency of the comK recombinant strain at the Amye site is obviously higher than that of the non-recombinant Bacillus subtilis BS168 standard strain, and the gel strip result also proves that the homologous recombination at the Amye site is successful (FIG. 8c, the fragment size is 1529 bp).
(2) Verification of the Upp site recombination function:
construction of a PMD19T-Upp-cm verification plasmid: designing primers with corresponding homologous sequences, and respectively amplifying to obtain an Upp upstream gene (Upp-up) amplified fragment, an Upp downstream gene (Upp-down) amplified fragment and a chloramphenicol (cm) gene amplified fragment.
The specific primer sequences are as follows:
in this example, the amplified fragment of the Upp upstream gene (Upp-up) was obtained by amplifying Bacillus subtilis BS168 genomic DNA as a template, wherein the amplification primers were:
upstream Upp 1-F: 5'-CGGTGAAGTATTGCAGGACG-3' (SEQ ID NO: 21);
downstream Upp 1-R: 5'-CCGTATATATGTCAGCTTGTGC-3' (SEQ ID NO: 22).
In this example, the amplified fragment of the Upp downstream gene (Upp-down) was obtained by amplifying Bacillus subtilis BS168 genomic DNA as a template, wherein the amplification primers were:
upstream Upp 2-F: 5'-GATATTTACATTGCGGCGCTAG-3' (SEQ ID NO: 23);
downstream Upp 2-R: 5'-CCATAACCCAGTACATACACTGCC-3' (SEQ ID NO: 24).
In this example, the amplified fragment of chloramphenicol (cm) gene was amplified using plasmid pHT254 as a template, wherein the amplification primers were:
upstream cm-F: 5'-ACAAGCTGACATATATACGGCGGCAATAGTTACCCTTATT-3' (SEQ ID NO: 25);
downstream cm-R: 5'-AGCGCCGCAATGTAAATATCTGTGGATAACCGTATTACCG-3' (SEQ ID NO: 26).
The three fragments were ligated by overlap PCR and ligated into the pMD19-T plasmid (purchased from Shanghai Baisai Biotechnology Ltd.) to obtain the pMD19T-Upp-cm plasmid,using this plasmid for BS168-P xylA -comK recombinant strain transformation, recombination verification. The method comprises the following specific steps: mu.L of pMD19T-Upp-cm plasmid with the concentration of 200 ng/mu.L is added into 100 mu.L of competent cell solution, the mixture is mixed evenly, incubated at 37 ℃ and 220r/min for 90min, coated on a cm (10 mu g/mL) resistant plate, inverted overnight at 37 ℃ for culture, and positive colonies are screened. As a control, a standard strain of non-recombinant Bacillus subtilis BS168 was used.
Meanwhile, verification of PCR amplification was performed using Upp1-F and Upp 2-R.
The results are shown in FIG. 9.
It can be found that BS168-P xylA The homologous recombination efficiency of the comK recombinant strain at the Upp site is obviously higher than that of the non-recombinant Bacillus subtilis BS168 standard strain, and the gel strip result also proves that the homologous recombination at the Upp site is successful (FIG. 9c, the fragment size is 2396 bp).
(3) And (3) verifying the transformation function of the plasmid:
in this example, the detection of BS168-P was performed using pHT blank 254 (derived from the above examples) and pHT blank 43 (available from Wuhan vast Ling Biotech Limited feeds) as test plasmids xylA Plasmid transformation capacity of comK recombinant strains.
The method comprises the following specific steps: mu.L of plasmid solution (blank pHT254 or blank pHT43) at a concentration of 200 ng/. mu.L was added to 100. mu.L of BS168-P xylA The comK recombinant strain (competent) solution is mixed uniformly, incubated at 37 ℃ for 90min at 220r/min, spread on a cm (10. mu.g/mL) resistant plate, inverted overnight at 37 ℃ for culture, and positive colonies are screened. As a control, a standard strain of non-recombinant Bacillus subtilis BS168 was used.
The results are shown in FIG. 10.
It can be found that BS168-P xylA The plasmid transformation capacity of the competent cells of the comK engineering strain is obviously higher than that of the non-recombinant Bacillus subtilis BS168 standard strain.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> southern China university of agriculture
<120> bacillus subtilis BS168 competence engineering strain and application thereof
<130>
<160> 26
<170> PatentIn version 3.5
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gaagtcctgc ggggaatggg caaattaagc gcagaggaaa gacctaagat gggcgccttg 180
gcaaacgaag tacgtgagag aatagcaaat gcgatagcag ataaaaacga gaagttggag 240
gaggaggaga tgaaacaaaa actggcaggt cagacaatag atgtaaccct tccgggcaac 300
cctgtggcag ttgggggacg ccatccgctc acggtggtca ttgaagagat agaagactta 360
ttcattggta tgggatatac agttgaagaa ggacctgaag tagaaacaga ttactataac 420
ttcgagagtc ttaatttacc aaaagagcat ccagcacgcg acatgcaaga ctccttctac 480
attacggaag agaccctcat gcgtacacag acaagtccag ttcagactcg cacaatggag 540
aaacacgaag gcaaaggccc ggtgaaaata atttgtccag gaaaagttta ccggcgggac 600
aatgatgatg caactcattc acatcagttt atgcagatcg agggattggt tgttgataaa 660
aacatctcaa tgagtgacct caagggtacc ttagaattag tggctaaaaa aatgtttgga 720
caggacagag agatccgcct gagaccatcc ttctttcctt ttactgagcc gtcagttgaa 780
gttgatgtga cttgcttcaa atgcggcggc aacggctgct cagtctgtaa aggcaccgga 840
tggatagaga ttttgggggc cggcatggta catccgaacg tgctgaaaat ggcagggttc 900
gatccgaaag agtatcaggg atttggcttt ggaatgggag tggagcgcat agctatgctg 960
aagtatggaa ttgatgatat tcgccatttc tatacgaatg acgtccgctt cattagtcag 1020
ttcaagcaag cttaa 1035
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence
<400> 8
ggaagggaat caaggagata aaag 24
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence
<400> 9
caggataaag cacagctaca gac 23
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence
<400> 10
atgagcgatg atgatatccg tt 22
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
tcacgaacga aaatcgccat 20
<210> 12
<211> 42
<212> DNA
<213> Artificial sequence
<400> 12
aatggcgatt ttcgttcgtg agcgatatcc acttcatcca ct 42
<210> 13
<211> 39
<212> DNA
<213> Artificial sequence
<400> 13
catattatgg cctccttaaa aataaattca ttcaaatac 39
<210> 14
<211> 42
<212> DNA
<213> Artificial sequence
<400> 14
tttttaagga ggccataata tgagtcagaa aacagacgca cc 42
<210> 15
<211> 41
<212> DNA
<213> Artificial sequence
<400> 15
aacggatatc atcatcgctc atctaatacc gttccccgag c 41
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence
<400> 16
cattgaatga cggggcagac 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence
<400> 17
gactcttcat catcattggc 20
<210> 18
<211> 19
<212> DNA
<213> Artificial sequence
<400> 18
gttgacgcgg tcatcaatc 19
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence
<400> 19
tgtccagcca tcacattgtg 20
<210> 20
<211> 2586
<212> DNA
<213> Artificial sequence
<400> 20
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gatgggatgt cacgcagaat 540
tcattgctcg ggctgtatga ctggaataca caaaatacac aagtacagtc ctatctgaaa 600
cggttcttag acagggcatt gaatgacggg gcagacggtt ttcgatttga tgccgccaaa 660
catatagagg cgatatccac ttcatccact ccatttgttt aatctttaaa ttaagtatca 720
acatagtaca tagcgaatct tccctttatt atatctaatg tgttcataaa aaactaaaaa 780
aaatattgaa aatactgacg aggttatata agatgaaaat aagttagttt gtttaaacaa 840
caaactaata ggtgatgtac ttactatatg aaataaaatg catctgtatt tgaatgaatt 900
tatttttaag gaggccataa tatgagtcag aaaacagacg cacctttaga atcgtatgaa 960
gtgaacggcg caacaattgc cgtgctgcca gaagaaatag acggcaaaat ctgttccaaa 1020
attattgaaa aagattgcgt gttttatgta aacatgaagc cgctgcaaat tgtcgacaga 1080
agctgccgat tttttggatc aagctatgcg ggaagaaaag caggaactta tgaagtgaca 1140
aaaatttcac acaagccgcc gatcatggtg gacccttcga accaaatctt tttattccct 1200
acactttctt cgacaagacc ccaatgcggc tggatttccc atgtgcatgt aaaagaattc 1260
aaagcgactg aattcgacga tacggaagtg acgttttcca atgggaaaac gatggagctg 1320
ccgatctctt ataattcgtt cgagaaccag gtataccgaa cagcgtggct cagaaccaaa 1380
ttccaagaca gaatcgacca ccgcgtgccg aaaagacagg aatttatgct gtacccgaaa 1440
gaagagcgga cgaagatgat ttatgatttt attttgcgtg agctcgggga acggtattag 1500
acggacaagc tagtgacatg ggtagagtcg catgatacgt atgccaatga tgatgaagag 1560
tcgacatgga tgagcgatga tgatatccgt ttaggctggg cggtgatagc ttctcgttca 1620
ggcagtacgc ctcttttctt ttccagacct gagggaggcg gaaatggtgt gaggttcccg 1680
gggaaaagcc aaataggcga tcgcgggagt gctttatttg aagatcaggc tatcactgcg 1740
gtcaatagat ttcacaatgt gatggctgga cagcctgagg aactctcgaa cccgaatgga 1800
aacaaccaga tatttatgaa tcagcgcggc tcacatggcg ttgtgctggc aaatgcaggt 1860
tcatcctctg tctctatcaa tacggcaaca aaattgcctg atggcaggta tgacaataaa 1920
gctggagcgg gttcatttca agtgaacgat ggtaaactga caggcacgat caatgccagg 1980
tctgtagctg tgctttatcc tgatgatatt gcaaaagcgc ctcatgtttt ccttgagaat 2040
tacaaaacag gtgtaacaca ttctttcaat gatcaactga cgattacctt gcgtgcagat 2100
gcgaatacaa caaaagccgt ttatcaaatc aataatggac cagagacggc gtttaaggat 2160
ggagatcaat tcacaatcgg aaaaggagat ccatttggca aaacatacac catcatgtta 2220
aaaggaacga acagtgatgg tgtaacgagg accgagaaat acagttttgt taaaagagat 2280
ccagcgtcgg ccaaaaccat cggctatcaa aatccgaatc attggagcca ggtaaatgct 2340
tatatctata aacatgatgg gagccgagta attgaattga ccggatcttg gcctggaaaa 2400
ccaatgacta aaaatgcaga cggaatttac acgctgacgc tgcctgcgga cacggataca 2460
accaacgcaa aagtgatttt taataatggc agcgcccaag tgcccggtca gaatcagcct 2520
ggctttgatt acgtgctaaa tggtttatat aatgactcgg gcttaagcgg ttctcttccc 2580
cattga 2586
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence
<400> 21
cggtgaagta ttgcaggacg 20
<210> 22
<211> 22
<212> DNA
<213> Artificial sequence
<400> 22
ccgtatatat gtcagcttgt gc 22
<210> 23
<211> 22
<212> DNA
<213> Artificial sequence
<400> 23
gatatttaca ttgcggcgct ag 22
<210> 24
<211> 24
<212> DNA
<213> Artificial sequence
<400> 24
ccataaccca gtacatacac tgcc 24
<210> 25
<211> 40
<212> DNA
<213> Artificial sequence
<400> 25
acaagctgac atatatacgg cggcaatagt tacccttatt 40
<210> 26
<211> 40
<212> DNA
<213> Artificial sequence
<400> 26
agcgccgcaa tgtaaatatc tgtggataac cgtattaccg 40
Claims (10)
1. Bacillus subtilis BS168-P xylA -comK recombinant strain, characterized in that said Bacillus subtilis BS168-P xylA -comK recombinant strain with P inserted in Amye site xylA -comK gene.
2. The Bacillus subtilis BS168-P of claim 1 xylA -comK recombinant strain, characterized in that said Bacillus subtilis BS168-P xylA The comK recombinant strain does not contain a resistance tag.
3. The Bacillus subtilis BS168-P of claim 1 xylA -comK recombinant strain, characterized in that said Bacillus subtilis BS168-P xylA The nucleotide sequence of the comK recombinant strain is shown as SEQ ID NO: shown at 20.
4. The Bacillus subtilis BS168-P according to any one of claims 1 to 3 xylA -a method for constructing a comK recombinant strain comprising the steps of:
(1) construction of pDG1730-P grac -Phesm recombinant plasmid, transforming Bacillus subtilis BS168 to obtain Bacillus subtilis BS 168-Spc-Phesm;
(2) construction of resistance tag-free pDG1730 (. DELTA.Spc) -P xylA -comK recombinant plasmid, transforming Bacillus subtilis BS 168-Spc-Phesm;
(3) screening the positive strain with the resistance of 4CP, namely the bacillus subtilis BS168-P xylA -comK recombinant strain.
5. The method of construction of claim 4, wherein the pDG1730-P grac -the nucleotide sequence of the Phesm gene in the Phesm recombinant plasmid is shown as SEQ ID NO: shown at 7.
6. The method of claim 4, wherein the resistance tag comprises an ampicillin resistance tag, a spectinomycin resistance tag, and a chloramphenicol resistance tag.
7. A cell product comprising the Bacillus subtilis BS168-P of any one of claims 1 to 3 xylA -comK recombinant strain.
8. Use of the cell product of claim 7 for the preparation of a food, pharmaceutical or cosmetic product.
9. The Bacillus subtilis BS168-P according to any one of claims 1 to 3 xylA Application of the comK recombinant strain in construction of a Bacillus subtilis recombinant strain.
10. The Bacillus subtilis BS168-P according to any one of claims 1 to 3 xylA -use of comK recombinant strains for the construction of plasmid transformation vectors.
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| US20020182734A1 (en) * | 2000-08-11 | 2002-12-05 | Diaz-Torres Maria R. | Bacillus transformation, transformants and mutant libraries |
| CN105112350A (en) * | 2015-09-06 | 2015-12-02 | 西北农林科技大学 | Building method and application of lactobacillus reuteri resistance-marker-free gene integration system |
| CN105861405A (en) * | 2016-05-06 | 2016-08-17 | 中国科学院上海高等研究院 | High-conversion-rate bacillus subtilis and structuring method thereof |
| CN109735480A (en) * | 2019-02-27 | 2019-05-10 | 光明乳业股份有限公司 | A kind of recombined bacillus subtilis synthesizing the new tetrose of lactoyl-N- and its construction method and application |
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| US20020182734A1 (en) * | 2000-08-11 | 2002-12-05 | Diaz-Torres Maria R. | Bacillus transformation, transformants and mutant libraries |
| CN105112350A (en) * | 2015-09-06 | 2015-12-02 | 西北农林科技大学 | Building method and application of lactobacillus reuteri resistance-marker-free gene integration system |
| CN105861405A (en) * | 2016-05-06 | 2016-08-17 | 中国科学院上海高等研究院 | High-conversion-rate bacillus subtilis and structuring method thereof |
| CN109735480A (en) * | 2019-02-27 | 2019-05-10 | 光明乳业股份有限公司 | A kind of recombined bacillus subtilis synthesizing the new tetrose of lactoyl-N- and its construction method and application |
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