WO2023240607A1 - 一种用于编辑可生成细菌纤维素细菌基因组的方法 - Google Patents

一种用于编辑可生成细菌纤维素细菌基因组的方法 Download PDF

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WO2023240607A1
WO2023240607A1 PCT/CN2022/099492 CN2022099492W WO2023240607A1 WO 2023240607 A1 WO2023240607 A1 WO 2023240607A1 CN 2022099492 W CN2022099492 W CN 2022099492W WO 2023240607 A1 WO2023240607 A1 WO 2023240607A1
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sequence
bacteria
homology arm
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高艳梅
李飞旋
金帆
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Shenzhen Institute of Advanced Technology of CAS
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/02—Acetobacter

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  • the present invention belongs to the field of biotechnology, and specifically relates to a method for rapid modification of bacterial genomes that can produce bacterial cellulose.
  • Gene modification is a common and mature technology for studying bacterial gene functions, regulating gene circuits, and screening specific phenotypes.
  • microorganisms that can produce natural bacterial cellulose, such as Acetobacter xylinum their growth rate is slow, and their culture methods and molecular biology operations are quite different from commonly used bacteria such as Escherichia coli, which can be used to edit their genomes.
  • Red recombination technology which has been relatively mature in bacteria such as Escherichia coli
  • bacteria that can produce bacterial cellulose, such as Acetobacter xylinum
  • the operation method is complicated and time-consuming.
  • the present invention provides a single recombination method. Plasmids carrying homologous fragments can be linearly inserted into specific genomic sites using only one allelic exchange. This method does not require auxiliary plasmids, requires less electroporation than the above-mentioned Red recombination method, and the plasmid used for recombination only requires a homologous arm at the target position. In other words, the method of the present invention is simple to operate and takes less time, especially for batch genotype and phenotype research work.
  • One aspect of the present invention provides a method for gene editing of bacteria that can produce cellulose membranes.
  • the method includes the following steps:
  • S1 Determine the target modified genes or sites that can produce cellulose membrane bacteria, and determine the first homology arm sequence for allelic exchange with the donor plasmid based on the target modified genes or sites;
  • the replication origin site in the donor plasmid is a replication origin site that cannot replicate autonomously in the cellulose membrane-producing bacterium;
  • the first homology arm fragment and the second homology arm sequence are fragments capable of allelic exchange in the bacterial genome that can produce cellulose membranes and in the donor plasmid respectively.
  • the second homology arm sequence The sequence should be completely identical to the DNA sequence of the first homology arm.
  • the gene editing methods include inactivation of the target gene, mutation of specific sites of the target gene, gene fusion expression at the end of the target gene, insertion of foreign genes at specific positions in the genome, and modification of targets in the genome of bacteria that can produce cellulose membranes individually or simultaneously. Gene promoters and RBS.
  • the gene editing method is to inactivate the target gene.
  • the first homology arm sequence is a part of the target gene to be inactivated.
  • the first homology arm sequence is located in the middle of the target gene to be inactivated. .
  • the gene editing method is specific site mutation of the target gene.
  • the first homology arm sequence is a partial sequence of the target gene or a partial sequence of the target gene and its continuous downstream sequence; wherein the target gene
  • the partial sequence is a continuous DNA sequence including the base sequence of the position to be mutated and the stop codon.
  • the position to be mutated is located in the middle or end of the first homology arm.
  • the second homology arm sequence The other base sequences are 100% homologous to the first homology arm.
  • the gene editing method is gene fusion expression at the end of the target gene; in step S1), the first homology arm sequence is the target gene and does not include the target gene stop codon, but includes the amino acid sequence encoding the stop codon. of continuous segments.
  • the preferred first homology arm sequence starts from any position that does not include the complete start codon sequence of the target gene and ends with the base encoding the amino acid before its stop codon; in step S2), the donor plasmid also contains The inserted foreign gene; the upstream of the start codon of the foreign gene to be inserted does not contain a promoter and RBS, the transcription direction is consistent with the transcription direction of the target gene, and optionally, the coding sequence of the amino acid chain for connection is included.
  • the gene editing method is the insertion of foreign genes at specific positions in the genome.
  • the first homology arm sequence is the site to be inserted and its continuous upstream sequence; in step S2), the donor The plasmid also contains the foreign gene to be inserted, which includes a promoter and RBS sequence before the start codon of the foreign gene. Furthermore, the first homology arm sequence is a partial sequence in the target gene to be inactivated.
  • the gene editing method is to modify the target gene promoter and RBS in the bacterial genome that can produce cellulose membranes individually or simultaneously.
  • the first homology arm is the part of the target gene starting from the start codon. sequence; in step S2), the donor plasmid also contains a replaced promoter and or RBS sequence. The replaced promoter and or RBS must be at the 5' end of the homology arm, and the transcription direction is consistent with the transcription direction of the target gene.
  • the second homology arm segment includes at least a part of the target modified gene, or is homologous to at least a part of the target modified gene, or at least a part of the second homology arm segment is selected from the group consisting of The target modified gene and its downstream partial sequence are homologous to the specific editing site and its upstream partial sequence.
  • the second homology arm fragment is selected from those homologous to the target modified gene and its downstream partial sequence, and homologous to the specific editing site and its upstream partial sequence
  • the basic homology is at least 90 More than % sequence homology, or at least 95% or more sequence homology, or at least 96% or more sequence homology, or at least 97% or more sequence homology, or at least 98% or more sequence homology, or at least 99 % or more of sequence homology; or the basic homology means homology to other bases except 1 base, homology to other bases except 2 bases, homology to other bases except 3 bases. All but 4 bases are homologous, and all but 5 bases are homologous.
  • the length of the first and or second homology arm segments is at least 10 bases, preferably 50-1000 bases, such as 50, 60, 70, 80, 90, 100, 200, 300, 500, 600, 700, 800, 900, 1000 bases.
  • step S3) also includes a step of detecting through polymerase chain reaction (PCR) and second-generation sequencing to determine whether the obtained gene editing results of bacteria that can produce cellulose membranes are accurate.
  • the polymerase chain reaction (PCR) and sequencing methods are to amplify the gene editing site sequence through upstream and downstream primers of the gene editing site, and obtain the gene editing site through sequencing methods, such as second-generation sequencing methods. sequence.
  • step S2 the method for preparing donor plasmid in step S2 is:
  • the donor plasmid also contains the new gene to be inserted into the bacterial genome, its promoter and ribosome binding site (RBS);
  • screening genes are antibiotic resistance genes, fluorescent protein genes, marker protein genes, and auxotrophic screening markers.
  • the antibiotic resistance gene is selected from the group consisting of blatem, blastv, blarob, blaoxa, blaZ, aadB, aacC1, aacC2, aacC3, aac6'-IIa, aacA4, aad(6'), vanA, vanB, vanC, msrA , satA, aac(6')-aph(2”), vat, vga, ermA, ermB, ermC, mecA, int and sul.
  • replication origin site that cannot replicate in bacteria that can produce cellulose membranes is selected from replication origins that can replicate in any bacteria except bacteria that can generate cellulose membranes.
  • any bacterium other than bacteria capable of producing cellulose membranes may be Escherichia coli.
  • replication origin sites pMB1, R6K, RK2, Pro1600, ColE1, and pBR322 that cannot be replicated in bacteria that can produce cellulose membranes.
  • the gene-edited sequence is a partial sequence of the coding region or non-coding region to be inactivated.
  • step S3 the method of transferring bacteria capable of producing cellulose membranes in step S3) is electrical transduction or chemical transduction.
  • the bacteria that can produce bacterial cellulose are selected from the group consisting of Acetobacter xylinum, Acetobacter pasteurianum, Acetobacter xylogluconic acid, Gluconacetobacter henselae, Acetobacter aceti, Acetobacter acetogenes, Aerobacter, Rhizobium At least one of Bacillus, Achromobacter, Agrobacterium, Pseudomonas, Alcaligenes, Sarcina, and Dynabacter.
  • the present invention found that by utilizing the principle of allelic exchange, when the donor plasmid and the bacterial genome have a certain length of homology arms, the donor plasmid can be integrated into the bacterial genome, and then the bacterial genome can be edited. Therefore, the homologous sequence of the donor plasmid must contain a DNA sequence homologous to the target gene or a specific site in the genome.
  • the homology arm When used for specific gene modification, the homology arm must and can contain part of the DNA sequence of the target gene; when editing a specific site in the genome, the homology arm consists of the specific site DNA sequence and its continuous upstream DNA of any length. sequence composition.
  • the donor plasmid is not completely consistent with the DNA sequence of the homology arm in the corresponding genome.
  • the corresponding single or multiple bases in the homology arm sequence in the donor plasmid must be determined by The original base is mutated into the designed base.
  • the present invention utilizes a single recombination method to achieve rapid and simple genetic modification of bacterial genomes that can produce cellulose membranes.
  • the operation is simple and takes a short time.
  • Figure 1 Schematic diagram of the single recombination method used in Example 1 for specific gene modification and gene insertion at specific sites in the Acetobacter xylinum genome.
  • A is the schematic diagram of inactivating specific genes in the bacterial genome that can produce cellulose membranes
  • B is the schematic diagram of inserting new genes into specific sites in the bacterial genome that can produce cellulose membranes or inactivating specific genes at the same time
  • C is the schematic diagram of The schematic diagram of the 3' end fusion of a specific gene in the bacterial genome that can produce cellulose membranes with foreign proteins
  • D is the schematic diagram of the target gene point or small segment mutation in the bacteria that can produce cellulose membranes
  • E is the schematic diagram of the ability to produce fibers by modification alone or simultaneously The schematic diagram of the target gene promoter and RBS in the membrane bacterial genome to adjust its expression level.
  • FIG. 2 Donor plasmid map of Example 1.
  • A is the donor plasmid map of pln2-acsC1’
  • B is the donor plasmid map of pln2-ccpAX’
  • C is the donor plasmid map of pln2-dgc2’.
  • FIG. 3 Example 1 PCR identification gel image and DNA sequencing results. Among them, A is the result of acsC1 group, B is the result of ccpAX’, and C is the result of dgc2’.
  • Figure 1A is a schematic diagram of the principle of the present invention for inactivating specific genes in the genome.
  • A1 Based on the experimental goals, determine the target inactivated genes (X) in the bacterial genome that can produce cellulose membranes.
  • the homology arm sequence (A') is then determined based on the target gene sequence.
  • A' is a partial sequence of the target inactivated gene X, preferably located in the middle of the target inactivated gene X.
  • the length of A' is at least 10 bases.
  • the sequence used as a homology arm in the genome X gene is also marked as A’, so the target modified gene X can be expressed as AA’A;
  • the bacteria that have not been successfully integrated will not survive when cultured in a medium containing antibiotics.
  • the successfully integrated bacteria will have resistance gene expression in the genome, so they can survive.
  • the recombinant bacteria were integrated after allelic exchange, so the sequence AA'A originally encoding the target protein X was divided into two sections, and the original protein could no longer be expressed, achieving the inactivation of the target gene.
  • Figure 1B is a schematic diagram of the principle of the present invention for inserting new genes into specific sites or inactivating specific genes in the genome of bacteria that can produce cellulose membranes.
  • the position and transcription of the new gene to be inserted on the vector plasmid are The direction is not required; if a new gene is inserted into a specific site in the genome, the homology arm A' must consist of the specific site and its upstream DNA sequence; if a new gene is inserted and a specific gene is inactivated at the same time, the homology arm sequence can only be composed of Partial sequence composition of the target gene.
  • the replication origin site on the plasmid backbone cannot replicate autonomously in bacteria with cellulose membranes. Screening genes are used to screen recombinant bacteria.
  • the bacteria that have not been successfully integrated will not survive when cultured in a medium containing antibiotics.
  • the successfully integrated bacteria will have resistance gene expression in the genome, so they can survive.
  • new foreign genes can be inserted into specific sites in the genome of the host bacteria or new genes can be inserted into the host genome while inactivating specific genes.
  • Figure 1C is a schematic diagram of the principle of fusion of a foreign protein at the 3' end of a specific gene in the bacterial genome that can produce cellulose membranes according to the present invention.
  • the target fusion gene X in the bacterial genome that can produce cellulose membranes. Then, based on the target fusion gene sequence, the homology arm sequence (A’) is determined. For ease of understanding, we label the sequence used in the homology arm in the genome as A’, so the target fusion gene X can be expressed as AA’.
  • a donor plasmid pln2-A’-B which contains a replication origin site, a screening gene, a gene to be inserted (B) and a homologous sequence (A’).
  • the gene to be inserted does not need to contain any promoter or RBS before the start codon of the foreign gene. It can contain a coding sequence for a short linker amino acid chain.
  • the transcription direction of the gene to be inserted must be the same as that of the target fusion gene.
  • the homology arm sequence in the donor plasmid can only consist of a partial DNA sequence of more than 10 bases upstream of the stop codon of the target fusion gene, and does not include its stop codon.
  • the replication origin site on the plasmid backbone cannot replicate autonomously in bacteria with cellulose membranes. Screening genes are used to screen recombinant bacteria.
  • Figure 1D is a schematic diagram of the principle of target gene point or small segment mutation of the present invention.
  • the target mutant genes in the bacterial genome that can produce cellulose membranes Based on the experimental goals, determine the target mutant genes in the bacterial genome that can produce cellulose membranes. Then, the homology arm sequence (mA) is determined based on the target mutated gene sequence and the planned mutation position. In order to facilitate understanding, the target mutant gene is X, and the sequence of the homology arm is marked as mA, so the target mutant gene X is expressed as AmA;
  • the homology arm mA is the partial DNA sequence of the target gene or the partial DNA sequence of the target modified gene and its continuous downstream DNA sequence.
  • the partial DNA sequence of the target gene must be a continuous DNA sequence including the base sequence at the mutation position and the stop codon.
  • the mutation position is located in the middle or end of the homology arm.
  • the other base sequences are not identical to the target gene.
  • the gene and its downstream sequences are 100% homologous.
  • the replication origin site on the plasmid backbone cannot replicate autonomously in bacteria with cellulose membranes. Screening genes are used to screen recombinant bacteria.
  • Figure 1E is a schematic diagram of the principle of the present invention by individually or simultaneously modifying the target gene promoter and RBS in the genome of a cellulose membrane-producing bacteria to adjust its expression level.
  • a donor plasmid pln2-P/R-A which contains a replication origin site, a screening gene, a replaced promoter (P) or RBS (R) and a homology arm sequence (tA).
  • the homology arm sequence must and can only consist of the partial DNA sequence of the X gene starting from the start codon.
  • the replaced promoter or RBS must be at the 5' end of the homology arm, and the direction is consistent with the transcription direction of the X gene; there is a replication origin site on the plasmid backbone that cannot initiate replication in bacteria that can produce cellulose membranes, and it must also Contains screening genes, which can be used to screen engineered bacteria after gene editing.
  • Acetobacter xylinum ATCC53582 strain was selected as the target strain, and the ccpAX, dgc2, and acsC1 genes in its genome were respectively inactivated as an example.
  • the vector plasmid pln2 used only contained the selection marker resistance gene aacC1 and the replication origin site pMB1.
  • the pMB1 origin of replication is replicable in E. coli but not in Acetobacter xylinum. Therefore, Acetobacter xylinum can only survive in the HS-resistant plate containing the corresponding resistance through allelic exchange with the homology arms in the donor plasmid and then integrating the entire vector plasmid backbone into its genome (Fig. 1 ).
  • the donor plasmids used in the experiment containing fragments of the ccpAX, dgc2, and acsC1 genes (ccpAX', dgc2', acsC1') were electroporated into Acetobacter xylinum ATCC53582, and then Bacteria use a single allelic exchange to destroy the target gene into two segments, thereby achieving gene inactivation.
  • Bacteria use a single allelic exchange to destroy the target gene into two segments, thereby achieving gene inactivation.
  • the method of the present invention successfully inactivated the ccpAX, dgc2, and acsC1 genes through a single recombination method ( Figure 3). It also confirmed that the single recombination technology can be used to generate cellulose membranes. Feasibility of bacterial genome modification.
  • E. coli TOP10 strains were used for plasmid construction, propagation, and preliminary screening of target plasmids.
  • Acetobacter xylinum ATCC58532 is the host strain.
  • LB broth was used as the growth medium for E. coli and Hestrin-Schramm (HS) medium was used for Acetobacter xylinum ATCC58532.
  • HS Hestrin-Schramm
  • the concentrations of gentamicin used are 15 ⁇ g/mL and 300 ⁇ g/mL for Escherichia coli and Acetobacter xylinum respectively.
  • the culture temperatures of Escherichia coli and Acetobacter xylinum were 37°C and 30°C, respectively.
  • Recombinant plasmid construction 1) First use PCR method to clone the target fragment (ccpAX', dgc2', acsC1') genes, and simultaneously PCR clone to prepare the linearized expression vector pln2; 2) Use Gibson assembly seamless connection technology, respectively Connect the target fragment (ccpAX', dgc2', acsC1') genes to the vector to construct a donor plasmid.
  • Single recombinant genome recombination operation steps 1) The plasmid after sequencing verification is electrotransduced into Acetobacter xylinum ATCC53582, and cultured in HS solid medium containing 300 ⁇ g/mL gentamicin; 2) Select a single clone point and use the corresponding primers Identify the genome insertion; 3) Cut the fragments that have been successfully inserted and send them for sequencing.

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Abstract

提供一种用于可生成细菌纤维素细菌基因组的基因编辑方法，所述方法包括以下步骤:S1)确定可生成纤维素膜细菌中的目标修改的基因或位点，并根据目标修改的基因或位点确定第一同源臂序列；S2)制备供体质粒，所述供体质粒包含复制起始位点、筛选基因以及用于和基因组等位交换的第二同源臂序列；S3)将制备获得的供体质粒转入可生成纤维素膜细菌中，进行培养，并通过与所述筛选基因对应的筛选方法进行筛选，进而得到基因编辑的可生成纤维素膜细菌的重组菌；所述重组菌基因组中的目标修改的基因或位点插入了线性化的供体质粒序列。本发明利用单重组的方法，对可生成纤维素膜的细菌基因组快速、简便的基因修改。

Description

一种用于编辑可生成细菌纤维素细菌基因组的方法 技术领域
本发明属于生物技术领域，具体涉及一种用于可生成细菌纤维素细菌基因组的快速修改的方法。
背景技术
基因修改是研究细菌基因功能、调控基因回路、筛选特定表型等方面一种常见而成熟的技术。然而，对于可生成天然细菌纤维素细菌，例如木醋杆菌等微生物来说，其生长速度较慢、培养方式和分子生物学操作与常用的大肠杆菌等细菌相差较大、可用于编辑其基因组的技术目前只有Red重组技术被报道。
目前虽然有研究者利用Red重组技术实现了对木醋杆菌基因组的敲除，但这一敲除首先需要将辅助质粒(helper plasimde)电转入野生型木醋杆菌中，同时还需要通过重叠聚合酶链式反应(overlap PCR)将靶标基因上下游同源臂和抗性基因共三个片段构建成一个重组片段；其次需要再一次电转将得到的重组片段电转入含有辅助质粒的木醋杆菌中；最后若需要获得纯净敲除，需要经历一次额外电转、一次抗生素培养基筛选。这种策略虽然很经典但操作步骤多，流程复杂，尤其是对于生长周期较慢的木醋杆菌等微生物，每多经历一次电转无疑是要多花4-5天的时间。
因此，在大肠杆菌等细菌中已经较为成熟的Red重组技术虽然可以在可生成细菌纤维素细菌，例如木醋杆菌中操作，但是却存在操作方法复杂，耗时太长的问题。这些因素均很大程度的限制了研究者对它们基因组基因和表型的研究以及工程化这类细菌。
发明内容
因此，为了缩短对可生成纤维素膜且生长速度较慢的木醋杆菌等细菌基因重组(敲除，插入，修改，蛋白融合等操作)时间，本发明提供了一种单次重组的方法，实现仅利用一次等位基因交换就可以将携带同源片段的质粒线性化插入特定的基因组位点。这种方法不需要辅助质粒，比上述Red重组方法少一次电转，并且用于重组的质粒只需要目标位置一段同源臂即可。换言之本发明的方法操作简单且耗时较短，尤其对于批量的基因型与表型研究工作来说。
本发明一个方面提供了一种可生成纤维素膜细菌基因编辑方法，所述方法包括以下步骤：
S1)确定可生成纤维素膜细菌中的目标修改的基因或位点，并根据目标修改的基因或位点 确定用于和供体质粒进行等位交换的第一同源臂序列；
S2)制备供体质粒，所述供体质粒包含复制起始位点、筛选基因以及用于和可生成纤维素膜细菌基因组中第一同源臂序列进行等位交换的第二同源臂序列；
S3)将制备获得的供体质粒转入可生成纤维素膜细菌中，进行培养，并通过与所述筛选基因对应的筛选方法进行筛选，进而得到基因编辑的可生成纤维素膜细菌的重组菌；所述重组菌基因组的目标修改的基因或位点插入了线性化的供体质粒序列；
在供体质粒中的复制起始位点为在所述可生成纤维素膜细菌中无法自主复制的复制起始位点；
所述第一同源臂片段和第二同源臂序列分别为可生成纤维素膜细菌基因组以及供体质粒中能够进行等位交换的片段，除同于目标基因突变外，第二同源臂序列应与第一同源臂DNA序列完全一致。
进一步地，所述基因编辑方法为目标基因失活、目标基因特定位点突变、目标基因末尾基因融合表达、基因组特定位置外源基因的插入、单独或同时修改可生成纤维素膜细菌基因组中目标基因启动子和RBS。
进一步地，所述基因编辑方法为目标基因失活，在步骤S1)中，第一同源臂序列为待失活目标基因的一部分，优选第一同源臂序列为位于待失活目标基因中间。
进一步地，所述基因编辑方法为目标基因特定位点突变，在步骤S1)中，第一同源臂序列为目标基因部分序列或者目标基因部分序列和其连续的下游序列；其中所述目标基因部分序列为包含待突变位置碱基序列和终止密码子的连续DNA序列，优选地，待突变位置位于第一同源臂中间或者末端，同时除待突变位置碱基外，第二同源臂序列中其它碱基序列与第一同源臂100％同源。
进一步地，所述基因编辑方法为目标基因末尾基因融合表达；在步骤S1)中，第一同源臂序列为目标基因且不包含目标基因终止密码子，但包含编码终止密码子前一个氨基酸序列的连续片段。优选的第一同源臂序列从不包含目标基因完整起始密码子序列的任意位置起始到编码其终止密码子前一个氨基酸的碱基终止；在步骤S2)中，供体质粒还包含待插入的外源基因；待插入基因的外源基因起始密码子上游不包含启动子和RBS，转录方向与目标基因转录方向一致，任选地，包含连接用的氨基酸链的编码序列。
进一步地，所述基因编辑方法为基因组特定位置外源基因的插入，在步骤S1)中，第一同源臂序列为待插入位点及其连续的上游序列；在步骤S2)中，供体质粒还包含待插入的外源基因，所述外源基因起始密码子前包含启动子和RBS序列。更进一步地，第一同源臂序列为 待失活的目标基因中的部分序列。
进一步地，所述基因编辑方法为单独或同时修改可生成纤维素膜细菌基因组中目标基因启动子和RBS，在步骤S1)中，第一同源臂为目标基因自起始密码子开始的部分序列；在步骤S2)中，供体质粒还包含替换的启动子和或RBS序列，替换的启动子和或RBS必须在同源臂的5’端，且转录方向与目标基因转录方向一致。
进一步地，所述第二同源臂片段包含了目标修改的基因中的至少一部分，或者与目标修改的基因中的至少一部分同源，或者所述第二同源臂片段中至少一部分选自与目标修改的基因及其下游部分序列同源，与特定编辑位点及其上游部分序列同源。
进一步地，所述第二同源臂片段中至少一部分选自与目标修改的基因及其下游部分序列同源，与特定编辑位点及其上游部分序列同源，所述基本同源为至少90％以上的序列同源，或至少95％以上的序列同源，或至少96％以上的序列同源，或至少97％以上的序列同源，或至少98％以上的序列同源，或至少99％以上的序列同源；或者所述基本同源为除1个碱基以外其他碱基同源，除2个碱基以外其他碱基同源，除3个碱基以外其他碱基同源除4个碱基以外同源，其他碱基同源除5个碱基以外其他碱基同源。
进一步地，第一和或第二同源臂片段的长度为至少10个碱基，优选为50-1000个碱基，例如50、60、70、80、90、100、200、300、500、600、700、800、900、1000个碱基。
进一步地，在步骤S3)后还包含通过聚合酶链式反应(PCR)以及二代测序进行检测的步骤，已确定得到的可生成纤维素膜细菌的基因编辑结果是否准确。更进一步地，所述聚合酶链式反应(PCR)以及测序的方法为通过基因编辑位点上下游引物扩增基因编辑位点序列，并通过测序方法，例如二代测序方法获得基因编辑位点序列。
进一步地，所述重组菌基因组中插入了线性化的供体质粒序列。
进一步地，步骤S2)中制备供体质粒的方法为：
S2-1)获得编辑包含目标的基因或位点的DNA序列的同源片段；
S2-2)将用于编辑目标的基因或位点的DNA序列的同源片段与表达载体质粒连接获得供体质粒，所述供体质粒包含在可生成纤维素膜细菌中不可复制的复制起始位点和筛选基因；
进一步地，所述供体质粒还含有待插入细菌基因组的新基因及其启动子和核糖体结合位点(RBS)；
进一步地，所述筛选基因为抗生素抗性基因、荧光蛋白基因、标记蛋白基因，营养缺陷型筛选标记。
进一步地，所述的抗生素抗性基因选自blatem、blashv、blarob、blaoxa、blaZ、aadB、aacC1、 aacC2、aacC3、aac6’－IIa、aacA4、aad(6’)、vanA、vanB、vanC、msrA、satA、aac(6’)－aph(2”)、vat、vga、ermA、ermB、ermC、mecA、int和sul。
进一步地，所述在可生成纤维素膜细菌中不可复制的复制起始位点选自在除可生成纤维素膜细菌外任意一种细菌中可以复制的复制起点。例如，除可生成纤维素膜细菌外任意一种细菌可以为大肠杆菌。
进一步地，所述在可生成纤维素膜细菌中不可复制的复制起始位点pMB1、R6K、RK2、Pro1600、ColE1、pBR322。
进一步地，基因编辑的序列为待失活的编码区或者非编码区的部分序列。
进一步地，步骤S3)中转入可生成纤维素膜细菌的方法为电转导或化学转导。
进一步地，所述可生成细菌纤维素细菌选自木醋杆菌、巴氏醋杆菌、木葡糖酸醋杆菌、汉氏葡糖醋杆菌、醋化醋杆菌、产醋醋杆菌、气杆菌、根瘤杆菌、无色杆菌、土壤杆菌、假单胞杆菌、产碱杆菌、八叠球菌、动胶菌中的至少一种。
本发明发现，利用等位交换的原理，当供体质粒和细菌基因组有一定长度同源臂就可以将供体质粒整合到其基因组中，进而对细菌基因组进行编辑。因此供体质粒的同源序列中必须包含目标基因或基因组特定位点同源的DNA序列。当用于特定基因修改时，同源臂必须且可以包含目标基因的部分DNA序列；当同于编辑基因组特定位点时，此同源序列由特定位点DNA序列和其连续的上游任意长度DNA序列组成。此外，若对基因组某一特定基因进行突变时，供体质粒与其对应的基因组中同源臂DNA序列不完全一致，供体质粒中的同源臂序列中对应位置单个或多个碱基要由原始碱基突变成设计的碱基。
有益效果
本发明利用单重组的方法，实现对可生成纤维素膜的细菌基因组快速，简便的基因修改。操作简单，用时短。
附图说明
图1：实施例1中单重组方法用于木醋杆菌基因组特定基因修改和特定位点基因插入的原理图。
其中，A为可生成纤维素膜细菌基因组中特定基因失活的原理图，B为向可生成纤维素膜细菌基因组中特定位点或失活特定基因的同时插入新基因的原理图，C为可生成纤维素膜细菌基因组中特定基因3’端融合外源蛋白的原理图，D为可生成纤维素膜细菌中目标基因 点或小段突变的原理图，E为通过单独或同时修改可生成纤维素膜细菌基因组中目标基因启动子和RBS进而调整其表达量的原理图。
图2：实施例1供体质粒图谱。其中A为pln2-acsC1’的供体质粒图谱，B为pln2-ccpAX’的供体质粒图谱,C为pln2-dgc2’的供体质粒图谱。
图3：实施例1PCR鉴定胶图以及DNA测序结果。其中A为acsC1组的结果，B为ccpAX’的结果，C为dgc2’的结果。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
结合图1的A说明可生成纤维素膜细菌基因组中特定基因失活的原理。图1A为本发明用于失活基因组中特定基因原理的示意图。
S1)根据实验目标，确定可生成纤维素膜细菌基因组中的目标失活基因(X)。随后根据目标基因序列确定同源臂序列(A’)，A’为目标失活基因X的部分序列，优选为位于目标失活基因X中间，A’的长度为至少10个碱基。为了方便理解，将基因组X基因中用作同源臂的序列也标为A’，因此目标修改基因X可表示为AA’A；
S2)制备供体质粒Pln2-A’，所述供体质粒包含复制起始位点、筛选基因以及同源臂序列(A’)；其中同源臂序列只能由目标基因A的部分序列组成。在质粒骨架上的复制起始位点无法在具有在可生成纤维素膜细菌中自主复制。筛选基因是为了对重组菌进行筛选。
S3)将制备获得的供体质粒转入可生成纤维素膜细菌中，进行培养，由于供体质粒中的复制起始位点无法在可生成纤维素膜细菌中自主复制，只能通过同源序列A’与基因组进行等位交换将质粒中的基因全部整合到可生成纤维素膜细菌的基因组中才能被复制。同时由于质粒中包含了筛选基因，通过与所述筛选基因对应的筛选方法进行筛选，就可得到特定基因失活的可生成纤维素膜细菌的重组菌。例如当选择的筛选基因为抗生素抗性基因时，采用含有抗生素的培养基培养时未整合成功的细菌则无法存活，整合成功得到重组菌基因组中具有抗性基因表达，因此可以存活。同时重组菌由于等位交换后进行了整合，因此将原编码目标蛋白X的序列AA’A分成了两段，无法再表达原蛋白，实现了目标基因的失活。
结合图1的B说明向可生成纤维素膜细菌基因组中特定位点或失活特定基因的同时插入新基因的原理。图1B为本发明向可生成纤维素膜细菌基因组中特定位点或失活特定基因的同时插入新基因的原理示意图。
S1)根据实验目标，确定可生成纤维素膜细菌基因组中的目标插入位点或者基因。随后根据目标插入位点或者基因序列，确定同源臂序列(A’)。
S2)制备供体质粒pln2-B-A’，所述供体质粒包含复制起始位点、筛选基因、待插入的新基因(B)和同源序列(A’)。其中待插入基因组的新基因B起始密码子前需要包含启动子和RBS序列，启动子和RBS根据实验中计划外源基因表达方式和强度决定，待插入的新基因在载体质粒上位置和转录方向不做要求；若将新基因插入基因组特定位点，同源臂A’必须由特定位点及其上游DNA序列组成；若插入新基因同时失活特定基因，其中同源臂序列只能由目标基因的部分序列组成。在质粒骨架上的复制起始位点无法在具有在可生成纤维素膜细菌中自主复制。筛选基因是为了对重组菌进行筛选。
S3)将制备获得的供体质粒转入可生成纤维素膜细菌中，进行培养，由于供体质粒中的起始位点无法在可生成纤维素膜细菌中进行自主复制，只能通过同源的序列A’进行等位交换将质粒中的基因全部整合到可生成纤维素膜细菌的基因组中才能复制，由于质粒中包含了筛选基因，通过与所述筛选基因对应的筛选方法进行筛选，就可得到在特定基因组位点或者基因中插入新基因的可生成纤维素膜细菌的重组菌。例如当选择的筛选基因为抗生素抗性基因时，采用含有抗生素的培养基培养时未整合成功的细菌则无法存活，整合成功得到重组菌基因组中具有抗性基因表达，因此可以存活。该重组菌通过等位交换后，即可将新的外源基因插入宿主菌基因组中特定位点或者在失活特定基因的同时插入新的基因到宿主基因组中。
结合图1的C说明可生成纤维素膜细菌基因组中特定基因3’端融合外源蛋白的原理。图1C为本发明可生成纤维素膜细菌基因组中特定基因3’端融合外源蛋白的原理示意图。
S1)根据实验目标，确定可生成纤维素膜细菌基因组中的目标融合基因X。随后根据目标融合基因序列，确定同源臂序列(A’)。为了方便理解，我们将基因组中用在同源臂的序列标为A’，因此目标融合基因X可表示为AA’.
S2)制备供体质粒pln2-A’-B，所述供体质粒包含复制起始位点、筛选基因、待插入基因(B)和同源序列(A’)。其中待插入基因的外源基因起始密码子前不需要包含任何启动子和RBS，可包含小段连接用的氨基酸链(linker)的编码序列，待插入基因的转录方向必须与目标融合基因相同。供体质粒中同源臂序列只能由目标融合基因终止密码子上游10个以上碱基长度的部分DNA序列组成，且不包含其终止密码子。在质粒骨架上的复制起始位点无法在具有在可生成纤维素膜细菌中自主复制。筛选基因是为了对重组菌进行筛选。
S3)将制备获得的供体质粒转入可生成纤维素膜细菌中，进行培养，由于供体质粒中的起始位点无法在可生成纤维素膜细菌中进行自主复制，只能通过同源的序列A’进行等位交换 将质粒中的基因全部整合到可生成纤维素膜细菌的基因组中才能复制，由于质粒中包含了筛选基因，通过与所述筛选基因对应的筛选方法进行筛选，进而得到基因组中特定基因融合表达的重组菌。例如当选择的筛选基因为抗生素抗性基因时，采用含有抗生素的培养基培养时未整合成功的细菌则无法存活，整合成功得到重组菌基因组中具有抗性基因表达，因此可以存活。该重组菌通过等为交换后，即可将新的外源基因与宿主菌基因组中特定基因进行融合。
结合图1D说明可生成纤维素膜细菌中目标基因点或小段突变的原理。图1D为本发明目标基因点或小段突变原理示意图。
S1)根据实验目标，确定可生成纤维素膜细菌基因组中的目标突变基因。随后根据目标突变基因序列以及计划突变位置，确定同源臂序列(mA)。为了方便理解，目标突变基因为X，同源臂的序列标为mA，因此目标突变基因X表示为AmA；
S2)制备供体质粒pln-mA，所述供体质粒包含复制起始位点、筛选基因，以及同源臂(mA)。其中同源臂mA为目标基因部分DNA序列或者目标修改基因部分DNA序列和其连续的下游DNA序列。其中目标基因的部分DNA序列必须是包含突变位置碱基序列和终止密码子的连续DNA序列，优选地突变位置位于同源臂中间或者末端，同时除突变位置碱基外，其它碱基序列与目标基因及其下游序列100％同源。在质粒骨架上的复制起始位点无法在具有在可生成纤维素膜细菌中自主复制。筛选基因是为了对重组菌进行筛选。
S3)将制备获得的供体质粒转入可生成纤维素膜细菌中，进行培养，由于供体质粒中的起始位点无法在可生成纤维素膜细菌中进行起始复制表达，只能通过同源的序列进行等位交换将质粒中的基因全部整合到可生成纤维素膜细菌的基因组中，由于质粒中包含了筛选基因，通过与所述筛选基因对应的筛选方法进行筛选，进而得到基因编辑的可生成纤维素膜细菌重组菌。例如当选择的筛选基因为抗生素抗性基因时，采用含有抗生素的培养基培养时未整合成功的细菌则无法存活，整合成功得到重组菌基因组中具有抗性基因表达，因此可以存活。该细菌由于等位交换后进行了整合，即可将基因组中的目标基因的进行突变。
结合图1E说明通过单独或同时修改可生成纤维素膜细菌基因组中目标基因启动子和RBS进而调整其表达量的原理。图1E为本发明通过单独或同时修改可生成纤维素膜细菌基因组中目标基因启动子和RBS进而调整其表达量的原理示意图。
S1)根据实验目标，确定可生成纤维素膜细菌基因组中的目标突变基因X。随后根据目标基因，确定同源臂序列(tA)。为了方便区分和理解将基因组中X基因中用作同源臂的序列标为tA，即图中目标突变基因X以tAA行式展现；
S2)制备供体质粒pln2-P/R-A，所述供体质粒包含复制起始位点、筛选基因、替换的启动 子(P)或RBS(R)和同源臂序列(tA)。其中同源臂序列必须且只能由X基因从起始密码子开始的部分DNA序列组成。替换的启动子或RBS必须在同源臂的5’端，且方向与X基因转录方向一致；在质粒骨架上具有在可生成纤维素膜细菌中无法起始复制的复制起始位点，还包含筛选基因，筛选基因可以用于基因编辑后工程菌的筛选。
S3)将制备获得的供体质粒转入可生成纤维素膜细菌中，进行培养，由于供体质粒中的起始位点无法在可生成纤维素膜细菌中进行自主复制，只能通过同源臂进行等位交换将质粒中的基因全部整合到可生成纤维素膜细菌的基因组中才能复制，由于质粒中包含了筛选基因，通过与所述筛选基因对应的筛选方法进行筛选，进而得到基因编辑的可生成纤维素膜细菌重组菌。例如当选择的筛选基因为抗生素抗性基因时，采用含有抗生素的培养基培养时未整合成功的细菌则无法存活，整合成功得到重组菌基因组中具有抗性基因表达，因此可以存活。该重组菌通过等为交换后，目标基因的启动子或RBS就可被更改，随即其表达量进行改变。
实施例1单重组方法使木醋杆菌基因失活实验
选用木醋杆菌ATCC53582菌株为目标菌株，以分别失活其基因组中ccpAX、dgc2、acsC1基因为例。
在进行基因重组实验时，使用的载体质粒pln2只包含筛选标记的抗性基因aacC1和复制起始位点pMB1。pMB1复制起始位点在大肠杆菌中可复制，但在木醋杆菌中不可复制。因此，木醋杆菌只能通过与供体质粒中的同源臂发生等位交换，进而将整个载体质粒骨架整合到其基因组中才能在含有对应抗性的HS抗性板中生存下来(图1)。
如图2所示，实验中使用的含有ccpAX、dgc2、acsC1基因自身一部分片段(ccpAX’、dgc2’、acsC1’)的供体质粒，通过将供体质粒分别电转到木醋杆菌ATCC53582中，随后细菌借助单次等位基因交换将目的基因破坏为两段，从而实现基因失活。通过PCR鉴定以及对对应位置基因序列测序分析也证实本发明方法通过单重组的方法成功灭活ccpAX、dgc2、acsC1基因(图3)，同时也证实了单次重组技术用于可生成纤维素膜的细菌基因组修改的可行性。
菌种和生长条件：大肠杆菌TOP10菌株用于质粒构建、增殖以及目标质粒的初步筛选。木醋杆菌ATCC58532为宿主菌株。在所有实验中，大肠杆菌均使用LB肉汤用作生长培养基，木醋杆菌ATCC58532均使用Hestrin-Schramm(HS)培养基用作生长培养基。如需要添加抗生素，对大肠杆菌和木醋杆菌，庆大霉素使用浓度分别为15μg/mL和300μg/mL。所有的实验中，如非特别说明大肠杆菌和木醋杆菌培养温度分别为37℃和30℃。
重组质粒构建：1)首先利用PCR方法克隆目标片段(ccpAX’、dgc2’、acsC1’)基因，同时PCR克隆制备线性化的表达载体pln2；2)通过吉布森(Gibson assembly)无缝连接技术，分别将目标片段(ccpAX’、dgc2’、acsC1’)基因连接到载体上，构建出供体质粒。
单重组基因组重组操作步骤：1)测序验证后的质粒电转入木醋杆菌ATCC53582中，在含有300μg/mL庆大霉素的HS固体培养基培养；2)挑选单克隆点，用对应的引物鉴定基因组插入情况；3)将鉴定插入成功的片段切胶送测序。

Claims (10)

  1. 一种可生成纤维素膜细菌的基因编辑方法，其特征在于，所述基因编辑方法包括以下步骤：
    S1)确定可生成纤维素膜细菌中的目标修改的基因或位点，并根据目标修改的基因或位点确定用于和供体质粒进行等位交换的第一同源臂序列；
    S2)制备供体质粒，所述供体质粒包含复制起始位点、筛选基因以及用于和可生成纤维素膜细菌基因组中第一同源臂序列进行等位交换的第二同源臂序列；
    S3)将制备获得的供体质粒转入可生成纤维素膜细菌中，进行培养，并通过与所述筛选基因对应的筛选方法进行筛选，进而得到基因编辑的可生成纤维素膜细菌的重组菌；所述重组菌基因组的目标修改的基因或位点插入了线性化的供体质粒序列；
    在供体质粒中的复制起始位点为在所述可生成纤维素膜细菌中无法自主复制的复制起始位点；
    所述第一同源臂片段和第二同源臂序列分别为可生成纤维素膜细菌基因组以及供体质粒中能够进行等位交换的片段，除同于目标基因突变外，第二同源臂序列应与第一同源臂DNA序列完全一致；
    优选地，基因编辑的序列为待失活的编码区或者非编码区的部分序列；
    优选地，所述可生成细菌纤维素细菌选自木醋杆菌、巴氏醋杆菌、木葡糖酸醋杆菌、汉氏葡糖醋杆菌、醋化醋杆菌、产醋醋杆菌、气杆菌、根瘤杆菌、无色杆菌、土壤杆菌、假单胞杆菌、产碱杆菌、八叠球菌、动胶菌中的至少一种。
  2. 根据权利要求1所述的基因编辑方法，其特征在于，所述基因编辑方法为目标基因失活、目标基因特定位点突变、目标基因末尾基因融合表达、基因组特定位置外源基因的插入、单独或同时修改可生成纤维素膜细菌基因组中目标基因启动子和RBS。
  3. 根据权利要求2所述的基因编辑方法，其特征在于，所述基因编辑方法为目标基因失活，在步骤S1)中，第一同源臂序列为待失活目标基因的一部分，优选第一同源臂序列为位于待失活目标基因中间；或者
    所述基因编辑方法为目标基因特定位点突变，在步骤S1)中，第一同源臂序列为目标基因部分序列或者目标基因部分序列和其连续的下游序列；其中所述目标基因部分序列为包含待突变位置碱基序列和终止密码子的连续DNA序列，优选地，待突变位置位于第一同源臂中间或者末端，同时除待突变位置碱基外，第二同源臂序列中其它碱基序列与第一同源臂100％ 同源；或者
    所述基因编辑方法为目标基因末尾基因融合表达；在步骤S1)中，第一同源臂序列为目标基因且不包含目标基因终止密码子，但包含编码终止密码子前一个氨基酸序列的连续片段。优选的第一同源臂序列从不包含目标基因完整起始密码子序列的任意位置起始到编码其终止密码子前一个氨基酸的碱基终止；在步骤S2)中，供体质粒还包含待插入的外源基因；待插入基因的外源基因起始密码子上游不包含启动子和RBS，转录方向与目标基因转录方向一致，任选地，包含连接用的氨基酸链的编码序列；或者
    所述基因编辑方法为基因组特定位置外源基因的插入，在步骤S1)中，在步骤S1)中，第一同源臂序列为待插入位点及其连续的上游序列；在步骤S2)中，供体质粒还包含待插入的外源基因，所述外源基因起始密码子前包含启动子和RBS序列。更进一步地，第一同源臂序列为待失活的目标基因中的部分序列；或者
    所述基因编辑方法为单独或同时修改可生成纤维素膜细菌基因组中目标基因启动子和RBS，在步骤S1)中，第一同源臂为目标基因自起始密码子开始的部分序列；在步骤S2)中，供体质粒还包含替换的启动子和或RBS序列，替换的启动子和或RBS必须在同源臂的5’端，且转录方向与目标基因转录方向一致。
  4. 根据权利要求1所述的基因编辑方法，其特征在于，所述第二同源臂片段包含了目标修改的基因中的至少一部分，或者与目标修改的基因中的至少一部分同源，或者所述第二同源臂片段中至少一部分选自与目标修改的基因及其下游部分序列同源，与特定编辑位点及其上游部分序列同源。
  5. 根据权利要求1所述的基因编辑方法，其特征在于，所述第二同源臂片段中至少一部分选自与目标修改的基因及其下游部分序列同源或基本同源，与特定编辑位点及其上游部分序列同源或基本同源，所述基本同源为至少90％以上的序列同源，或至少95％以上的序列同源，或至少96％以上的序列同源，或至少97％以上的序列同源，或至少98％以上的序列同源，或至少99％以上的序列同源；或者所述基本同源为除1个碱基以外其他碱基同源，除2个碱基以外其他碱基同源，除3个碱基以外其他碱基同源除4个碱基以外同源，其他碱基同源除5个碱基以外其他碱基同源。
  6. 根据权利要求1所述的基因编辑方法，其特征在于，第一和或第二同源臂片段的长度为至少10个碱基，优选为15-1000个碱基。
  7. 根据权利要求1所述的基因编辑方法，其特征在于，步骤S2)中制备供体质粒的方法为：
    S2-1)获得编辑包含目标的基因或位点的DNA序列的同源片段；
    S2-2)将用于编辑目标的基因或位点的DNA序列的同源片段与表达载体质粒连接获得供体质粒，所述供体质粒包含在可生成纤维素膜细菌中不可复制的复制起始位点和筛选基因；
    优选地，所述供体质粒还含有待插入细菌基因组的新基因及其启动子和核糖体结合位点(RBS)。
  8. 根据权利要求1所述的基因编辑方法，其特征在于，进一步地，所述筛选基因为抗生素抗性基因、荧光蛋白基因、标记蛋白基因，营养缺陷型筛选标记；
    优选地，所述的抗生素抗性基因选自blatem、blashv、blarob、blaoxa、blaZ、aadB、aacC1、aacC2、aacC3、aac6’－IIa、aacA4、aad(6’)、vanA、vanB、vanC、msrA、satA、aac(6’)－aph(2”)、vat、vga、ermA、ermB、ermC、mecA、int和sul。
  9. 根据权利要求1所述的基因编辑方法，其特征在于，所述在可生成纤维素膜细菌中不可复制的复制起始位点选自在除可生成纤维素膜细菌外任意一种细菌中可以复制的复制起点；
    优选地，所述在可生成纤维素膜细菌中不可复制的复制起始位点pMB1、R6K、RK2、Pro1600、ColE1、pBR322。
  10. 根据权利要求1所述的可生成纤维素膜细菌基因编辑方法，其特征在于，在步骤S3)后还包含通过聚合酶链式反应以及二代测序进行检测的步骤，以确定得到的可生成纤维素膜细菌的基因编辑结果是否准确；
    优选地，所述聚合酶链式反应以及测序的方法为通过基因编辑位点上下游引物扩增基因编辑位点序列，并通过测序方法获得基因编辑位点序列；
    优选地，步骤S3)中转入可生成纤维素膜细菌的方法为电转导或化学转导。
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