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WO2025108354A1 - Engineered escherichia coli strain, and preparation method and use therefor - Google Patents

Engineered escherichia coli strain, and preparation method and use therefor Download PDF

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WO2025108354A1
WO2025108354A1 PCT/CN2024/133414 CN2024133414W WO2025108354A1 WO 2025108354 A1 WO2025108354 A1 WO 2025108354A1 CN 2024133414 W CN2024133414 W CN 2024133414W WO 2025108354 A1 WO2025108354 A1 WO 2025108354A1
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escherichia coli
stbl2
recf
coli strain
polya
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Chinese (zh)
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胡勇
刘文山
宋文
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Shenzhen Rhegen Biotechnology Co Ltd
Wuhan Rhegen Biotechnology Co Ltd
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Shenzhen Rhegen Biotechnology Co Ltd
Wuhan Rhegen Biotechnology Co Ltd
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Definitions

  • E. coli fermentation is currently the main method for preparing plasmids.
  • homologous recombination usually occurs, resulting in the loss of some fragments on the plasmid or the insertion of additional fragments.
  • the main homologous recombination enzyme recA of E. coli is usually knocked out.
  • Conventional E. coli used for preparing plasmids such as DH5 ⁇ , JM109, TOP10, stbl2, etc., all contain recA defects.
  • polyA sequence is usually constructed onto the DNA template of the plasmid.
  • PolyA is a simple tandem repeat sequence. In Escherichia coli, simple tandem repeat sequences usually lose or increase, but the specific mechanism is not clear at present. Possible mechanisms include: homologous recombination, sliding of DNA polymerase during DNA replication, DNA mismatch repair, etc.
  • the most widely used method is to insert other base sequences in the middle of polyA to avoid excessively long polyA sequences, but after a long period of fermentation, there will still be a small amount of polyA missing, and it also limits the range of polyA sequences that can be selected.
  • CN115461463A discloses a genetically modified Escherichia coli strain that can increase the stability of polyA, and the modification method is to knock out the sbcC and/or sbcD genes related to DNA repair in Escherichia coli.
  • the literature (X. Pan, D. R. Leach, The roles of mutS, sbcCD and recA in the propagation of TGG repeats in Escherichia coli, Nucl. Acids Res. 28 (2000) 3178–3184) reported that knocking out sbcC and sbcD on the basis of knocking out recA had no effect on the (TGG)24 tandem repeat sequence.
  • One object of the present invention is to provide an engineered Escherichia coli strain.
  • Another object of the present invention is to provide the use of the engineered Escherichia coli strain in producing plasmids.
  • Another object of the present invention is to provide a method for preparing a plasmid using the engineered Escherichia coli strain.
  • the present invention genetically modifies Escherichia coli in terms of homologous recombination and DNA mismatch repair.
  • the present invention selects rarA, recE, recF, recO, recR, and recJ as targets for modification.
  • the present invention selects DNA polymerase II and DNA polymerase V involved in DNA error repair in Escherichia coli as targets for modification.
  • the modification target of the present invention is to make the target gene not expressed, expressed missing or inactive protein.
  • the modification method can be point mutation, deletion of part or the entire ORF reading frame, etc.
  • the present invention provides an engineered Escherichia coli strain, in which one or more of the following proteins are under-expressed or not expressed, or express missing or inactive proteins: rarA, recE, recF, recO, recR, recJ.
  • the engineered Escherichia coli strain of the present invention has low expression or no expression of recF and/or recJ protein, or expresses missing or inactive recF and/or recJ protein.
  • the coding sequence of recF and/or recJ in the genome of the engineered Escherichia coli strain of the present invention is completely or partially truncated or mutated, so that the strain recF and/or recJ protein is lowly expressed or not expressed, or expresses missing or inactive recF and/or recJ protein.
  • the engineered Escherichia coli strain of the present invention further has low expression or no expression of recE protein, or expresses a missing or inactive recE protein.
  • the engineered Escherichia coli strain of the present invention further has low expression or no expression of DNA polymerase V, or expresses missing or inactive DNA polymerase V.
  • the engineered Escherichia coli strain of the present invention further has non-defective expression of DNA polymerase II.
  • the engineered Escherichia coli strain of the present invention is an engineered DH5 ⁇ or stbl2 strain.
  • the present invention also provides a method for preparing the engineered Escherichia coli strain, which comprises:
  • the protein of the E. coli strain is lowly expressed or not expressed, or the protein is missing or inactive.
  • the present invention is based on Escherichia coli stbl2, and knocks out part or the entire ORF reading frame of its rarA, recE, recF, recO, recR or recJ gene, so that the strain does not express the corresponding protein, and prepares the engineered strains Stbl2- ⁇ rarA, Stbl2- ⁇ recE, Stbl2- ⁇ recF, Stbl2- ⁇ recJ, Stbl2- ⁇ recO, Stbl2- ⁇ recR.
  • the present invention is based on Escherichia coli stbl2, and part or all of the ORF reading frames of the recF and recJ genes are knocked out, so that the strain does not express the corresponding recF and recJ proteins, and the engineered strain Stbl2- ⁇ recF ⁇ recJ is prepared.
  • the present invention is based on Escherichia coli stbl2, and part or the entire ORF reading frame of its recE and recF genes is knocked out, so that the strain does not express the corresponding recE and recF proteins, and the engineered strain Stbl2- ⁇ recE ⁇ recF is prepared.
  • the present invention is based on Escherichia coli stbl2, and part or the entire ORF reading frame of its recE, recF and recJ genes is knocked out, so that the strain does not express the corresponding recE, recF and recJ proteins, and the engineered strain Stbl2- ⁇ recE ⁇ recF ⁇ recJ is prepared.
  • the present invention is based on Escherichia coli stbl2, and part or the entire ORF reading frame of its recE, recF and rarA genes is knocked out, so that the strain does not express the corresponding recE, recF and rarA proteins, and the engineered strain Stbl2- ⁇ recE ⁇ recF ⁇ rarA is prepared.
  • the present invention is based on Escherichia coli stbl2, and knocks out part or the entire ORF reading frame of its recE, recF and DNA polymerase pol V genes, so that the strain does not express the corresponding recE, recF proteins and DNA polymerase pol V, and prepares the engineered strain Stbl2- ⁇ recE ⁇ recF ⁇ polV.
  • the present invention is based on Escherichia coli stbl2, and knocks out part or the entire ORF reading frame of its recE, recF and DNA polymerase pol II genes, so that the strain does not express the corresponding recE, recF proteins and DNA polymerase pol II, and prepares the engineered strain Stbl2- ⁇ recE ⁇ recF ⁇ pol II.
  • the present invention is based on Escherichia coli stbl2, and knocks out part or the entire ORF reading frame of its recE, recF, DNA polymerase pol V and DNA polymerase pol II genes, so that the strain does not express the corresponding recE, recF proteins, DNA polymerase pol V and DNA polymerase pol II, and prepares the engineered strain Stbl2- ⁇ recE ⁇ recF ⁇ polV ⁇ polII.
  • the present invention is based on Escherichia coli DH5 ⁇ , knocks out part or all of the ORF reading frame of its recF gene, so that the strain does not express the corresponding recF protein, and prepares the engineered strain DH5 ⁇ - ⁇ recF.
  • the present invention is based on Escherichia coli DH5 ⁇ , knocks out part or all of the ORF reading frame of its recJ gene, so that the strain does not express the corresponding recJ protein, and prepares the engineered strain DH5 ⁇ - ⁇ recJ.
  • the present invention is based on Escherichia coli DH5 ⁇ , knocks out part or all of the ORF reading frames of its recF and recJ genes, so that the strain does not express the corresponding recF and recJ proteins, and prepares the engineered strain DH5 ⁇ - ⁇ recF ⁇ recJ.
  • the present invention also provides the use of the engineered Escherichia coli strain in producing plasmids.
  • the plasmid is a plasmid containing a tandem repeat sequence.
  • the tandem repeat sequence is a simple tandem repeat sequence, for example, a short sequence consisting of one or several relatively constant bases (e.g., 1-6 nt) as a repeating unit, connected end to end, and connected in series to form a repeating sequence.
  • the tandem repeat sequence includes but is not limited to a polyA sequence.
  • the present invention also provides a method for preparing a plasmid, in particular a plasmid containing a tandem repeat sequence (such as a polyA sequence), the method comprising:
  • the engineered Escherichia coli strain of the present invention is used to ferment and prepare a plasmid containing a tandem repeat sequence (eg, a polyA sequence).
  • a tandem repeat sequence eg, a polyA sequence
  • the tandem repeat sequence is a poly A sequence.
  • the poly A sequence contains at least 12 nt, at least 24 nt, at least 36 nt or at least 48 nt of base A and 0-24 nt or 0-12 nt of non-A bases.
  • the full length of the poly A sequence is 24 nt-144 nt, more preferably 90 nt-120 nt.
  • the fermentation conditions are: culturing at 28-32° C. for 12 h to 72 h.
  • the engineered Escherichia coli strain of the present invention ferments and prepares a plasmid containing a tandem repeat sequence, and has a certain plasmid production stability.
  • the engineered Escherichia coli strains of the present invention Stbl2- ⁇ rarA, Stbl2- ⁇ recF, Stbl2- ⁇ recJ, Stbl2- ⁇ recR, Stbl2- ⁇ recF ⁇ recJ, Stbl2- ⁇ recE ⁇ recF, Stbl2- ⁇ recE ⁇ recF ⁇ recJ, Stbl2- ⁇ recE ⁇ recF ⁇ polV, DH5 ⁇ - ⁇ recF, DH5 ⁇ - ⁇ recJ, DH5 ⁇ - ⁇ recF ⁇ recJ can improve the plasmid production stability to a certain extent.
  • FIG1 shows the results of PCR identification of knockout experiments of rarA.
  • FIG. 2 shows the experimental results of stability evaluation of polyA120 of the stbl2 strain.
  • FIG. 3 shows the experimental results of stability evaluation of polyA120 of the stbl2- ⁇ recE strain.
  • FIG. 4 shows the experimental results of stability evaluation of polyA120 of the stbl2- ⁇ recF strain.
  • FIG. 5 shows the experimental results of stability evaluation of polyA120 of the stbl2- ⁇ recE ⁇ recF ⁇ recJ strain.
  • the present invention genetically modifies Escherichia coli in terms of homologous recombination and DNA mismatch repair.
  • the present invention selects rarA, recE, recF, recO, recR, and recJ as targets for modification.
  • the present invention selects DNA polymerase II and DNA polymerase V involved in DNA error repair in Escherichia coli as targets for modification.
  • the modification target of the present invention is to make the target gene not expressed, expressed missing or inactive protein.
  • the modification method can be point mutation, deletion of part or the entire ORF reading frame, etc.
  • the vector pEcCas (addgene 73227) and pEcgRNA (addgene 166581) were used to knock out rarA in Escherichia coli stbl2 strain using CRISPR-Cas9.
  • the upstream homology arm of rar A was amplified by PCR
  • the downstream homology arm of rarA was amplified by PCR using primers CGCTCGATTTTTCTAATGTTATCGTTGCGGTAATGTTGTT (SEQ ID No.6) and GGGAGTTACGCTCCGCTTGC (SEQ ID No.7).
  • the two homology arms were spliced into the rarA knockout homology arm using overlapPCR. Gene knockout was performed according to the method described in the literature Q Li, B Sun, J Chen, et al.
  • a modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli Acta Biochim Biophys Sin, 2021, 53(5), 620–627, deleting the DNA fragment at positions 27-1341 of rarA (1344bp) on the genome.
  • the resulting sequence was GTGAGCAATCTGTCGCTCGATTTTTCTAA (SEQ ID No. 8).
  • the knockout steps are as follows:
  • PCR identification Randomly pick a single colony and perform colony PCR verification using primers CTTTGTCCTGACGCCGAAAGC (SEQ ID No. 4) and GGGAGTTACGCTCCGCTTGC (SEQ ID No. 7).
  • the size of the fragment with successful knockout should be 503 bp, and the size of the fragment with failed knockout should be 1818 bp.
  • the single colonies obtained from the above screening were inoculated into 3mL liquid LB containing 5g/L glucose and cultured overnight at 37°C and 220rpm for 16h.
  • the bacterial solution was then spread on an LB plate containing 5g/L glucose and 10g/l sucrose, and cultured overnight at 37°C for 16h.
  • Single colonies were randomly selected and plated on LB plates and LB plates containing 50 ⁇ g/ml kanamycin. Colonies sensitive to kanamycin successfully eliminated pEcCas and glycerol-protected bacteria.
  • a polyA of 120 A was inserted into the pUC18 plasmid, and the stability of polyA was verified using a plasmid containing 120 A (polyA120).
  • clones with the correct polyA size were screened by PCR. They were inoculated into LB liquid culture medium and cultured at 30°C for 20 hours, then spread on plates. 40 clones were randomly selected, and partial sequences containing polyA120 were amplified by PCR. The size of the PCR product was observed by agarose gel electrophoresis.
  • Example 2 Knockout of recE in Escherichia coli stbl2 and its evaluation
  • the DNA fragment at positions 47-288 of the recE gene (2601bp) on the stbl2 genome was deleted.
  • the sequence of the deleted fragment was: CCGGTGAACCTGACGTCGTCCTGTGGGCAAGCA ACGATTTTGAATCGACCTGTGCCACTCTGGACTACCTGATCGTTAAGTCAGGTAAAAAACTGAGCAGCTATTTTAAAGCTGTTGCCACGAATTTTCCTGTCGTTAATGACCTGCCCGCTGAAGGTGAGATCGATTTTACCTGGAGTGAACGCTATCAACTCAGCAAAGACTCCATGACATGGGAACTAAAACCGGGAGCAGCACCAGAC (SEQ ID No.9).
  • Example 3 Knockout of recF in Escherichia coli stbl2 and its evaluation
  • the DNA fragment at positions 347-621 of the recF gene (1074 bp) on the stbl2 genome was deleted.
  • the sequence of the deleted fragment was: TGATAACGCCAGAAGGGTTTACTTTACTCAACGGCGGCCCCAAATACAGAAGAGCATTCCTCGACTGGGGATGCTTTCACAACGAACCCGGATTTTTCACCGCCTGGAGCAATCTCAAGCGATTGCTCAAGCAGCGCAATGCGGCGCTGCGCCAGGTGACACGTTACGAACAGCTACGCCCGTGGGATAAAGAGCTGATCCCGCTGGCGGAGCAAATCAGCACCTGGCGCGCGGAGTATAGCGCCGGTATCGCGCGATATGGCTGATACCTGT (SEQ ID No.10).
  • Example 4 Knockout of recJ in Escherichia coli stbl2 and its evaluation
  • Example 5 Knockout of recO or recR in Escherichia coli stbl2 and its evaluation
  • the recO deletion fragment sequence is ATGGAAGGCTGGCAGCGCGCATTTGTCCTGCATAGTCGCCCGTGGAGCGAAACCAGCCTGATGCTGGACGTCTTCACGGAGGAATCGGGGCGCGTGCGTCTGGTTGCCAAAGGCGCACGCTCTAAACGCTCTACCCTGAAAGGTGCATTACAGCCTTTCACCCCTCTCTTGCTAC GTTTTGGCGGGCGTGGCGAAGTCAAAACGCTGCGCAGTGCTGAAGCCGTCTCGCTGGCGCTGCCATTAAGCGGTATCACGCTTTACAGCGGTCTGTACATCAACGAACTTCTCTCCCGCGTACTGGAATACGAGACGCGCTTCTCTGAACTTTTTTCGATTACTTGCACTGCATTCAGTCTCTTGCAGG GGTCACTGGTACGCCAGAACCCGCGCTGCCGCTTTGAACTGGCACTGCTCGGGCATCTGGGTTATGGCGTCAATTTTACCCATTGCGGGTAGCGGCGAGCCGGTAGATGACACCATGACGTATCGTTATCGTTATCGGGTTAAA
  • the sequence of the RecR deletion fragment is: ATGCAAACCAGCCCGCTGTTAACACAGCTTATGGAAGCACTGCGCTGTCTGCCGGGCGTTGGCCCGAAGTCGGCGCAGCGTATGGCGTTCACGCTGCTTCAGCGCGATCGTAGCGGCGGGATGCGTCTGGCGCAGGCTCACCC GGGCGATGTCGGAAATCGGCCACTGCGCCGATTGCCGCACTTTCACCGAACAGGAAGTCTGTAACATCTGTTCGAATCCGCGTCGTCAGGAAAACGGTCAAATCTGCGTGGTGGAGAGTCCGGCGGACATCTACGCCATTGAGCAGACGGGGCAGTTT TCAGGTCGTTATTTTGTGTTGATGGGGCATCTGTCACCGCTGGACGGCATCGGTCCGGATGATATCGGGCTTGATCGTCTGGAACAGCGTCTGGCAGAGGAAAAAATCACTGAAGTGATCCTCGCCACCAACCCCACGGTTGAAGGTGAAGCTACCG CTAACTACATTGCCGAGCTTTGCGCAATATGAAAAAAA
  • Plasmids containing polyA120 were used to verify the effects of stbl2- ⁇ recO and stbl2- ⁇ recR on polyA stability. The results showed that the proportions of clones with obvious polyA deletion were 52.8% (38/72) and 47.2% (34/72), respectively, which were close to the original strain of stbl2. It can be seen that knocking out recO or recR does not affect the stability of polyA.
  • Example 6 Knockout of recF and/or recJ in Escherichia coli DH5 ⁇ and evaluation thereof
  • Example 7 Knockout of recJ based on stbl2- ⁇ recF and performance evaluation thereof
  • Example 2 The same method as in Example 1 was used to knock out recJ on the basis of stbl2- ⁇ recF to construct the strain stbl2- ⁇ recF ⁇ recJ.
  • Example 8 Knockout of recF or further knockout of recJ based on stbl2- ⁇ recE and performance evaluation thereof
  • Example 2 The same method as in Example 1 was used to knock out recF on the basis of stbl2- ⁇ recE to construct the strain stbl2- ⁇ recE ⁇ recF, and then on the basis of this strain, recJ was further knocked out to construct the strain stbl2- ⁇ recE ⁇ recF ⁇ recJ.
  • Example 9 Knockout of rarA based on stbl2- ⁇ recE ⁇ recF and performance evaluation thereof
  • Example 2 The same method as in Example 1 was used to knock out rarA on the basis of stbl2- ⁇ recE ⁇ recF to construct the strain stbl2- ⁇ recE ⁇ recF ⁇ rarA, and the effect of the strain on polyA stability was evaluated using polyA120. The results showed that the proportion of obvious polyA deletion was 55%, which was higher than that of stbl2- ⁇ recE ⁇ recF. Knocking out rarA did not further improve the stability of polyA120 of stbl2- ⁇ recE ⁇ recF.
  • Example 10 Knockout of DNA polymerase based on stbl2- ⁇ recE ⁇ recF and its evaluation
  • Example 2 The same method as in Example 1 was used to knock out the error-prone DNA polymerase pol II (polB, 2352 bp) and DNA polymerase pol V (1269 bp) separately and simultaneously on the basis of stbl2- ⁇ recE ⁇ recF, wherein pol II deleted the DNA fragment at positions 17-747, whose sequence was: TTATCTTAACCCGACACTGGCGGGACACCCCGCAAGGGACAGAAGTCTCCTTCTGGCTGGCGACGGACAACGGGCCGTTGCAGGTTACGCTTGCACCGCAAGAGTCCGTGGCGTTTATTCCCGCCGATCAGGTTCCCCGCGCTCAGCATATTTTGCAGGGTGAACAAGGCTTTCGCCTGACACCGCTGGCGTTAAAGGATTTTCACCGCCAGCCGGTGTATGGCCTTTACTGTCGCGCCCATCGCCAATTGATGAATTACGAAAAGCGCCTGCGTGAAGGTGGCGTTACCGTCTACGAGGCCGATGTGCGTCCGCCAGAAC GCTATCT
  • PolyA120 was also used to evaluate the effect of strains on polyA stability. The results showed that the proportions of obvious polyA deletion in these three strains were 52.5%, 30% and 60%, respectively, which were all higher than 12.5% of stbl2- ⁇ recE ⁇ recF. This shows that knocking out error-prone DNA polymerases cannot effectively improve the stability of polyA.
  • Example 11 Verification of the stability of polyA in stbl2- ⁇ recF and stbl2- ⁇ recE ⁇ recF using polyA90
  • Plasmids containing 90 consecutive A were introduced into stbl2- ⁇ recF and stbl2- ⁇ recE ⁇ recF, respectively. After culturing at 30°C for 20 hours, LB plates were coated and 72 monoclonal PCR fragments containing polyA90 were selected. The band size was identified by agarose gel electrophoresis. The proportions of stbl2, stbl2- ⁇ recF and stbl2- ⁇ recE ⁇ recF with obvious polyA deletion were 2.8%, 4.2% and 2.8%, respectively. Since the tandem repeat sequence of polyA90 is short, it is relatively stable under 30°C culture conditions, and there is no significant difference between different host bacteria.
  • Plasmids containing 90 consecutive A's were introduced into stbl2- ⁇ recF and stbl2- ⁇ recE ⁇ recF, respectively. After culture at 37°C for 20 h, the plates were spread on LB plates. Clones were randomly selected and PCR amplified for fragments containing polyA90. The band sizes were identified by agarose gel electrophoresis. The proportions of stbl2, stbl2- ⁇ recF and stbl2- ⁇ recE ⁇ recF with obvious polyA deletion were 94.4%, 91.7% and 58.3%, respectively.
  • Example 12 Verification of the stability of polyA in stbl2, stbl2- ⁇ recF, and stbl2- ⁇ recE ⁇ recF using multi-segmented polyA
  • the cells were spread on LB solid plates, and 96 single clones were selected for PCR verification of the polyA deletion rate.
  • the results showed that the proportions of clones with obvious deletions in stbl2, stbl2- ⁇ recF, and stbl2- ⁇ recE ⁇ recF were 21.9%, 6.3%, and 18.9%, respectively.

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Abstract

An engineered Escherichia coli strain, and a preparation method and use therefor. The engineered Escherichia coli strain has low expression or non-expression of recF and/or recJ protein, or expression of missing or inactive recF and/or recJ protein. Further, same has low expression of recE protein or non-expression, or expression of missing or inactive recE protein. The present invention can improve the stability of preparing a plasmid containing a polyA sequence using Escherichia coli.

Description

工程化大肠杆菌菌株及其制备方法与应用Engineered Escherichia coli strains and preparation methods and applications thereof 技术领域Technical Field

本发明是关于一种工程化大肠杆菌菌株及其制备方法与应用，具体而言，是关于一种可改善质粒生产稳定性的工程化大肠杆菌菌株、其制备方法以及在制备含有串联重复序列例如polyA序列的质粒中的应用。The present invention relates to an engineered Escherichia coli strain and a preparation method and application thereof, and in particular, to an engineered Escherichia coli strain capable of improving the stability of plasmid production, a preparation method thereof, and application thereof in preparing a plasmid containing a tandem repeat sequence such as a polyA sequence.

背景技术Background Art

大肠杆菌发酵是目前制备质粒的主要方法，当质粒上存在同源片段时，通常会发生同源重组，从而造成质粒上部分片段的丢失或额外片段的插入。为了防止同源重组，通常会将大肠杆菌的主要同源重组酶recA敲除，常规的用于制备质粒的大肠杆菌如DH5α、JM109、TOP10、stbl2等均含有recA缺陷。E. coli fermentation is currently the main method for preparing plasmids. When homologous fragments exist on the plasmid, homologous recombination usually occurs, resulting in the loss of some fragments on the plasmid or the insertion of additional fragments. In order to prevent homologous recombination, the main homologous recombination enzyme recA of E. coli is usually knocked out. Conventional E. coli used for preparing plasmids, such as DH5α, JM109, TOP10, stbl2, etc., all contain recA defects.

随着基因治疗和核酸疫苗领域的快速发展，对质粒的稳定性提出了更高的需求。在mRNA相关领域，为了增加mRNA的翻译，需要在其3’端添加较长的polyA序列，为了保证polyA序列的一致性，通常将polyA序列构建到质粒的DNA模板上。polyA属于简单串联重复序列，在大肠杆菌中，简单串联重复序列通常会发生缺失或增加的现象，但其具体的机制目前尚不明确，可能的机制包括：同源重组，DNA复制过程中DNA聚合酶的滑动，DNA的错配修复等。With the rapid development of gene therapy and nucleic acid vaccines, higher requirements are placed on the stability of plasmids. In the field of mRNA, in order to increase the translation of mRNA, a longer polyA sequence needs to be added to its 3' end. In order to ensure the consistency of the polyA sequence, the polyA sequence is usually constructed onto the DNA template of the plasmid. PolyA is a simple tandem repeat sequence. In Escherichia coli, simple tandem repeat sequences usually lose or increase, but the specific mechanism is not clear at present. Possible mechanisms include: homologous recombination, sliding of DNA polymerase during DNA replication, DNA mismatch repair, etc.

利用常规的recA缺陷的大肠杆菌如DH5α制备含有长的polyA的质粒时，polyA的缺失现象非常严重。可见polyA的缺失不依赖于recA，存在其他的未知途径。When conventional recA-deficient E. coli, such as DH5α, are used to prepare plasmids containing long polyA, the loss of polyA is very serious. This shows that the loss of polyA is not dependent on recA, but there are other unknown pathways.

为了保证polyA的稳定性，目前采用的技术手段主要集中在质粒的改造上，如使用线性质粒pVEL(A.E.Grier,S.Burleigh,J.Sahni,et al.pEVL:A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A)Sequences.Molecular Therapy-Nucleic Acids.2016,5:e306.)，但该方法质粒产量较低，目前没有广泛使用。目前使用较广泛的方法是在polyA中间插入其他的碱基序列，从而避免过长的polyA序列，但经过长时间的发酵还是会存在少量的polyA缺失的现象，此外还限制了polyA序列的可选择范围。In order to ensure the stability of polyA, the current technical means mainly focus on the transformation of plasmids, such as the use of linear plasmid pVEL (A.E.Grier, S.Burleigh, J.Sahni, et al. pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly (A) Sequences. Molecular Therapy-Nucleic Acids. 2016, 5: e306.), but this method has a low plasmid yield and is not widely used. The most widely used method is to insert other base sequences in the middle of polyA to avoid excessively long polyA sequences, but after a long period of fermentation, there will still be a small amount of polyA missing, and it also limits the range of polyA sequences that can be selected.

CN115461463A公开了一种遗传改造的大肠杆菌菌株可以增加polyA的稳定性，其改造方法是敲除了大肠杆菌中与DNA修复相关的sbcC和/或sbcD基因。而文献(X.Pan,D.R.Leach,The roles of mutS,sbcCD and recA in the propagation of TGG repeats in Escherichia coli,Nucl.Acids Res.28(2000)3178–3184)报道在recA敲除的基础上敲除sbcC和sbcD对(TGG)24串联重复序列无影响。CN115461463A discloses a genetically modified Escherichia coli strain that can increase the stability of polyA, and the modification method is to knock out the sbcC and/or sbcD genes related to DNA repair in Escherichia coli. However, the literature (X. Pan, D. R. Leach, The roles of mutS, sbcCD and recA in the propagation of TGG repeats in Escherichia coli, Nucl. Acids Res. 28 (2000) 3178–3184) reported that knocking out sbcC and sbcD on the basis of knocking out recA had no effect on the (TGG)24 tandem repeat sequence.

目前市场上并没有可以明显保证质粒polyA稳定性的大肠杆菌菌株。There is currently no E. coli strain on the market that can clearly ensure the stability of plasmid polyA.

发明内容Summary of the invention

本发明的一个目的在于提供一种工程化大肠杆菌菌株。One object of the present invention is to provide an engineered Escherichia coli strain.

本发明的另一目的在于提供所述的工程化大肠杆菌菌株在生产质粒中的应用。Another object of the present invention is to provide the use of the engineered Escherichia coli strain in producing plasmids.

本发明的另一目的在于提供利用所述工程化大肠杆菌菌株制备质粒的方法。Another object of the present invention is to provide a method for preparing a plasmid using the engineered Escherichia coli strain.

为了增加大肠杆菌生产质粒特别是生产含有串联重复序列例如polyA序列的质粒的稳定性，本发明针对同源重组、DNA错配修复两方面对大肠杆菌进行遗传改造。对于同源重组，本发明选择rarA，recE，recF，recO，recR，recJ作为目标进行改造。对于DNA错配修复，本发明选择大肠杆菌中参与DNA错误修复的DNA聚合酶II和DNA聚合酶V作为目标进行改造。本发明改造目标是使目标基因不表达、表达缺失或无活性的蛋白。改造方法可以是点突变、删除部分或整个ORF阅读框等。In order to increase the stability of Escherichia coli production plasmids, especially the production of plasmids containing tandem repeat sequences such as polyA sequences, the present invention genetically modifies Escherichia coli in terms of homologous recombination and DNA mismatch repair. For homologous recombination, the present invention selects rarA, recE, recF, recO, recR, and recJ as targets for modification. For DNA mismatch repair, the present invention selects DNA polymerase II and DNA polymerase V involved in DNA error repair in Escherichia coli as targets for modification. The modification target of the present invention is to make the target gene not expressed, expressed missing or inactive protein. The modification method can be point mutation, deletion of part or the entire ORF reading frame, etc.

一方面，本发明提供了一种工程化大肠杆菌菌株，其以下蛋白中的一种或多种蛋白低表达或不表达，或者表达缺失或无活性的蛋白：rarA，recE，recF，recO，recR，recJ。In one aspect, the present invention provides an engineered Escherichia coli strain, in which one or more of the following proteins are under-expressed or not expressed, or express missing or inactive proteins: rarA, recE, recF, recO, recR, recJ.

根据本发明的优选实施方案，本发明的工程化大肠杆菌菌株，其recF和/或recJ蛋白低表达或不表达，或者表达缺失或无活性的recF和/或recJ蛋白。According to a preferred embodiment of the present invention, the engineered Escherichia coli strain of the present invention has low expression or no expression of recF and/or recJ protein, or expresses missing or inactive recF and/or recJ protein.

根据本发明的具体实施方案，本发明的工程化大肠杆菌菌株，其基因组中的recF和/或recJ的编码序列被全部或部分地截短或者被突变，使得所述菌株recF和/或recJ蛋白低表达或不表达，或者表达缺失或无活性的recF和/或recJ蛋白。According to a specific embodiment of the present invention, the coding sequence of recF and/or recJ in the genome of the engineered Escherichia coli strain of the present invention is completely or partially truncated or mutated, so that the strain recF and/or recJ protein is lowly expressed or not expressed, or expresses missing or inactive recF and/or recJ protein.

根据本发明的具体实施方案，本发明的工程化大肠杆菌菌株，进一步地，其recE蛋白低表达或不表达，或者表达缺失或无活性的recE蛋白。According to a specific embodiment of the present invention, the engineered Escherichia coli strain of the present invention further has low expression or no expression of recE protein, or expresses a missing or inactive recE protein.

根据本发明的具体实施方案，本发明的工程化大肠杆菌菌株，进一步地，其DNA聚合酶V低表达或不表达，或者表达缺失或无活性的DNA聚合酶V。According to a specific embodiment of the present invention, the engineered Escherichia coli strain of the present invention further has low expression or no expression of DNA polymerase V, or expresses missing or inactive DNA polymerase V.

根据本发明的具体实施方案，本发明的工程化大肠杆菌菌株，进一步地，其DNA聚合酶II无缺陷表达。According to a specific embodiment of the present invention, the engineered Escherichia coli strain of the present invention further has non-defective expression of DNA polymerase II.

根据本发明的具体实施方案，本发明的工程化大肠杆菌菌株，其为工程化的DH5α或stbl2菌株。According to a specific embodiment of the present invention, the engineered Escherichia coli strain of the present invention is an engineered DH5α or stbl2 strain.

另一方面，本发明还提供了所述的工程化大肠杆菌菌株的制备方法，其包括：On the other hand, the present invention also provides a method for preparing the engineered Escherichia coli strain, which comprises:

采用点突变、删除部分或整个ORF阅读框的方法，使得大肠杆菌菌株的所述蛋白低表达或不表达，或者表达缺失或无活性的所述蛋白。By using point mutation or deleting part or the whole ORF reading frame, the protein of the E. coli strain is lowly expressed or not expressed, or the protein is missing or inactive.

在本发明的一些具体实施方案中，本发明以大肠杆菌stbl2为基础，分别敲除了其rarA、recE、recF、recO、recR或recJ基因的部分或整个ORF阅读框，使得菌株不表达相应蛋白，制备得到工程菌株Stbl2-ΔrarA、Stbl2-ΔrecE、Stbl2-ΔrecF、Stbl2-ΔrecJ、Stbl2-ΔrecO、Stbl2-ΔrecR。In some specific embodiments of the present invention, the present invention is based on Escherichia coli stbl2, and knocks out part or the entire ORF reading frame of its rarA, recE, recF, recO, recR or recJ gene, so that the strain does not express the corresponding protein, and prepares the engineered strains Stbl2-ΔrarA, Stbl2-ΔrecE, Stbl2-ΔrecF, Stbl2-ΔrecJ, Stbl2-ΔrecO, Stbl2-ΔrecR.

在本发明的一些具体实施方案中，本发明以大肠杆菌stbl2为基础，敲除了其recF和recJ基因的部分或整个ORF阅读框，使得菌株不表达相应recF和recJ蛋白，制备得到工程菌株Stbl2-ΔrecFΔrecJ。In some specific embodiments of the present invention, the present invention is based on Escherichia coli stbl2, and part or all of the ORF reading frames of the recF and recJ genes are knocked out, so that the strain does not express the corresponding recF and recJ proteins, and the engineered strain Stbl2-ΔrecFΔrecJ is prepared.

在本发明的一些具体实施方案中，本发明以大肠杆菌stbl2为基础，敲除了其recE和recF基因的部分或整个ORF阅读框，使得菌株不表达相应recE和recF蛋白，制备得到工程菌株Stbl2-ΔrecEΔrecF。In some specific embodiments of the present invention, the present invention is based on Escherichia coli stbl2, and part or the entire ORF reading frame of its recE and recF genes is knocked out, so that the strain does not express the corresponding recE and recF proteins, and the engineered strain Stbl2-ΔrecEΔrecF is prepared.

在本发明的一些具体实施方案中，本发明以大肠杆菌stbl2为基础，敲除了其recE、recF和recJ基因的部分或整个ORF阅读框，使得菌株不表达相应recE、recF和recJ蛋白，制备得到工程菌株Stbl2-ΔrecEΔrecFΔrecJ。In some specific embodiments of the present invention, the present invention is based on Escherichia coli stbl2, and part or the entire ORF reading frame of its recE, recF and recJ genes is knocked out, so that the strain does not express the corresponding recE, recF and recJ proteins, and the engineered strain Stbl2-ΔrecEΔrecFΔrecJ is prepared.

在本发明的一些具体实施方案中，本发明以大肠杆菌stbl2为基础，敲除了其recE、recF和rarA基因的部分或整个ORF阅读框，使得菌株不表达相应recE、recF和rarA蛋白，制备得到工程菌株Stbl2-ΔrecEΔrecFΔrarA。In some specific embodiments of the present invention, the present invention is based on Escherichia coli stbl2, and part or the entire ORF reading frame of its recE, recF and rarA genes is knocked out, so that the strain does not express the corresponding recE, recF and rarA proteins, and the engineered strain Stbl2-ΔrecEΔrecFΔrarA is prepared.

在本发明的一些具体实施方案中，本发明以大肠杆菌stbl2为基础，敲除了其recE、recF和DNA聚合酶pol V基因的部分或整个ORF阅读框，使得菌株不表达相应recE、recF蛋白和DNA聚合酶pol V，制备得到工程菌株Stbl2-ΔrecEΔrecFΔpolⅤ。In some specific embodiments of the present invention, the present invention is based on Escherichia coli stbl2, and knocks out part or the entire ORF reading frame of its recE, recF and DNA polymerase pol V genes, so that the strain does not express the corresponding recE, recF proteins and DNA polymerase pol V, and prepares the engineered strain Stbl2-ΔrecEΔrecFΔpolⅤ.

在本发明的一些具体实施方案中，本发明以大肠杆菌stbl2为基础，敲除了其recE、recF和DNA聚合酶pol II基因的部分或整个ORF阅读框，使得菌株不表达相应recE、recF蛋白和DNA聚合酶pol II，制备得到工程菌株Stbl2-ΔrecEΔrecFΔpol II。In some specific embodiments of the present invention, the present invention is based on Escherichia coli stbl2, and knocks out part or the entire ORF reading frame of its recE, recF and DNA polymerase pol II genes, so that the strain does not express the corresponding recE, recF proteins and DNA polymerase pol II, and prepares the engineered strain Stbl2-ΔrecEΔrecFΔpol II.

在本发明的一些具体实施方案中，本发明以大肠杆菌stbl2为基础，敲除了其recE、recF、DNA聚合酶pol V和DNA聚合酶pol II基因的部分或整个ORF阅读框，使得菌株不表达相应recE、recF蛋白、DNA聚合酶pol V和DNA聚合酶pol II，制备得到工程菌株Stbl2-ΔrecEΔrecFΔpolⅤΔpol Ⅱ。In some specific embodiments of the present invention, the present invention is based on Escherichia coli stbl2, and knocks out part or the entire ORF reading frame of its recE, recF, DNA polymerase pol V and DNA polymerase pol II genes, so that the strain does not express the corresponding recE, recF proteins, DNA polymerase pol V and DNA polymerase pol II, and prepares the engineered strain Stbl2-ΔrecEΔrecFΔpolⅤΔpolⅡ.

在本发明的一些具体实施方案中，本发明以大肠杆菌DH5α为基础，敲除了其recF基因的部分或整个ORF阅读框，使得菌株不表达相应recF蛋白，制备得到工程菌株DH5α-ΔrecF。In some specific embodiments of the present invention, the present invention is based on Escherichia coli DH5α, knocks out part or all of the ORF reading frame of its recF gene, so that the strain does not express the corresponding recF protein, and prepares the engineered strain DH5α-ΔrecF.

在本发明的一些具体实施方案中，本发明以大肠杆菌DH5α为基础，敲除了其recJ基因的部分或整个ORF阅读框，使得菌株不表达相应recJ蛋白，制备得到工程菌株DH5α-ΔrecJ。In some specific embodiments of the present invention, the present invention is based on Escherichia coli DH5α, knocks out part or all of the ORF reading frame of its recJ gene, so that the strain does not express the corresponding recJ protein, and prepares the engineered strain DH5α-ΔrecJ.

在本发明的一些具体实施方案中，本发明以大肠杆菌DH5α为基础，敲除了其recF和recJ基因的部分或整个ORF阅读框，使得菌株不表达相应recF和recJ蛋白，制备得到工程菌株DH5α-ΔrecFΔrecJ。In some specific embodiments of the present invention, the present invention is based on Escherichia coli DH5α, knocks out part or all of the ORF reading frames of its recF and recJ genes, so that the strain does not express the corresponding recF and recJ proteins, and prepares the engineered strain DH5α-ΔrecFΔrecJ.

另一方面，本发明还提供了所述的工程化大肠杆菌菌株在生产质粒中的应用。On the other hand, the present invention also provides the use of the engineered Escherichia coli strain in producing plasmids.

根据本发明的具体实施方案，所述质粒为含有串联重复序列的质粒。优选地，所述串联重复序列为简单串联重复序列，例如以相对恒定的一个或几个碱基组成的短序列(例如1-6nt)为重复单位，首尾相接，串联连接形成的重复序列。在本发明的一些具体实施方案中，所述串联重复序列包括但不限于polyA序列。According to a specific embodiment of the present invention, the plasmid is a plasmid containing a tandem repeat sequence. Preferably, the tandem repeat sequence is a simple tandem repeat sequence, for example, a short sequence consisting of one or several relatively constant bases (e.g., 1-6 nt) as a repeating unit, connected end to end, and connected in series to form a repeating sequence. In some specific embodiments of the present invention, the tandem repeat sequence includes but is not limited to a polyA sequence.

另一方面，本发明还提供了一种制备质粒特别是含有串联重复序列(例如polyA序列)的质粒的方法，该方法包括：On the other hand, the present invention also provides a method for preparing a plasmid, in particular a plasmid containing a tandem repeat sequence (such as a polyA sequence), the method comprising:

采用本发明所述的工程化大肠杆菌菌株发酵制备含有串联重复序列(例如polyA序列)的质粒。The engineered Escherichia coli strain of the present invention is used to ferment and prepare a plasmid containing a tandem repeat sequence (eg, a polyA sequence).

在本发明的一些具体实施方案中，所述串联重复序列为polyA序列。优选地，所述polyA序列含有至少12nt、至少24nt、至少36nt或至少48nt的碱基A以及0-24nt或0-12nt的非A碱基。根据本发明的优选实施方案，所述polyA序列的全长为24nt-144nt，更优选为90nt-120nt。In some specific embodiments of the present invention, the tandem repeat sequence is a poly A sequence. Preferably, the poly A sequence contains at least 12 nt, at least 24 nt, at least 36 nt or at least 48 nt of base A and 0-24 nt or 0-12 nt of non-A bases. According to a preferred embodiment of the present invention, the full length of the poly A sequence is 24 nt-144 nt, more preferably 90 nt-120 nt.

在本发明的一些具体实施方案中，本发明的制备质粒的方法中，所述发酵条件为：28-32℃培养12h-72h。In some specific embodiments of the present invention, in the method for preparing a plasmid of the present invention, the fermentation conditions are: culturing at 28-32° C. for 12 h to 72 h.

本发明所述的工程化大肠杆菌菌株发酵制备含有串联重复序列的质粒，具有一定的质粒生产稳定性。在本发明的一些具体实施方案中，本发明的工程化大肠杆菌菌株Stbl2-ΔrarA、Stbl2-ΔrecF、Stbl2-ΔrecJ、Stbl2-ΔrecR、Stbl2-ΔrecFΔrecJ、Stbl2-ΔrecEΔrecF、Stbl2-ΔrecEΔrecFΔrecJ、Stbl2-ΔrecEΔrecFΔpolⅤ、DH5α-ΔrecF、DH5α-ΔrecJ、DH5α-ΔrecFΔrecJ均可一定程度改善质粒生产稳定性。The engineered Escherichia coli strain of the present invention ferments and prepares a plasmid containing a tandem repeat sequence, and has a certain plasmid production stability. In some specific embodiments of the present invention, the engineered Escherichia coli strains of the present invention Stbl2-ΔrarA, Stbl2-ΔrecF, Stbl2-ΔrecJ, Stbl2-ΔrecR, Stbl2-ΔrecFΔrecJ, Stbl2-ΔrecEΔrecF, Stbl2-ΔrecEΔrecFΔrecJ, Stbl2-ΔrecEΔrecFΔpolV, DH5α-ΔrecF, DH5α-ΔrecJ, DH5α-ΔrecFΔrecJ can improve the plasmid production stability to a certain extent.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1显示PCR鉴定rarA的敲除实验结果。FIG1 shows the results of PCR identification of knockout experiments of rarA.

图2显示stbl2菌株polyA120的稳定性评价实验结果。FIG. 2 shows the experimental results of stability evaluation of polyA120 of the stbl2 strain.

图3显示stbl2-ΔrecE菌株polyA120的稳定性评价实验结果。FIG. 3 shows the experimental results of stability evaluation of polyA120 of the stbl2-ΔrecE strain.

图4显示stbl2-ΔrecF菌株polyA120的稳定性评价实验结果。FIG. 4 shows the experimental results of stability evaluation of polyA120 of the stbl2-ΔrecF strain.

图5显示stbl2-ΔrecEΔrecFΔrecJ菌株polyA120的稳定性评价实验结果。FIG. 5 shows the experimental results of stability evaluation of polyA120 of the stbl2-ΔrecEΔrecFΔrecJ strain.

具体实施方式DETAILED DESCRIPTION

为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。本发明实施例中所用起始试剂材料均为商购来源。各实施例中未注明具体条件的实验方法，按照所属领域的常规条件或按照厂商说明书建议的条件进行操作。In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of the present invention. The starting reagents and materials used in the examples of the present invention are all commercially available. The experimental methods in the examples without specifying specific conditions are operated according to the conventional conditions in the field or the conditions recommended by the manufacturer's instructions.

为了增加大肠杆菌polyA的稳定性，本发明针对同源重组和DNA错配修复两个方面对大肠杆菌进行遗传改造。对于同源重组，本发明选择rarA，recE，recF，recO，recR，recJ作为目标进行改造。对于DNA错配修复，本发明选择大肠杆菌中参与DNA错误修复的DNA聚合酶II和DNA聚合酶V作为目标进行改造。本发明改造目标是使目标基因不表达、表达缺失或无活性的蛋白。改造方法可以是点突变、删除部分或整个ORF阅读框等。In order to increase the stability of Escherichia coli polyA, the present invention genetically modifies Escherichia coli in terms of homologous recombination and DNA mismatch repair. For homologous recombination, the present invention selects rarA, recE, recF, recO, recR, and recJ as targets for modification. For DNA mismatch repair, the present invention selects DNA polymerase II and DNA polymerase V involved in DNA error repair in Escherichia coli as targets for modification. The modification target of the present invention is to make the target gene not expressed, expressed missing or inactive protein. The modification method can be point mutation, deletion of part or the entire ORF reading frame, etc.

实施例1、大肠杆菌stbl2中rarA基因的敲除及其评价Example 1. Knockout of the rarA gene in Escherichia coli stbl2 and its evaluation

本实施例使用载体pEcCas(addgene 73227)和pEcgRNA(addgene 166581)，采用CRISPR-Cas9的方法在大肠杆菌stbl2菌株的基础上敲除rarA。选择rarA的编辑位点GGATAATACTTTTCAACCTC(SEQ ID No.1)，利用引物TAGTGGATAATACTTTT CAACCTC(SEQ ID No.2)和AAACGAGGTTGAAAAGTATTATCC(SEQ ID No.3)退火后插入pEcgRNA的BsaI位点取代原来的2个BsaI位点之间的片段，构建pEcgRN A-rarA载体。利用引物CTTTGTCCTGACGCCGAAAGC(SEQ ID No.4)和CCGCA ACGATAACATTAGAAAAATCGAGCGACAGATTGC(SEQ ID No.5)，PCR扩增rar A上游同源臂，利用引物CGCTCGATTTTTCTAATGTTATCGTTGCGGTAATGTTGTT(SEQ ID No.6)和GGGAGTTACGCTCCGCTTGC(SEQ ID No.7)，PCR扩增rarA下游同源臂，将2条同源臂利用overlapPCR拼接成rarA敲除同源臂。按照文献Q Li,B Sun,J Chen,et al.A modified pCas/pTargetF system for CRISPR-Cas9-assisted gen ome editing in Escherichia coli.Acta Biochim Biophys Sin,2021,53(5),620–627记载的方法进行基因敲除，删除基因组上rarA(1344bp)的27-1341位的DNA片段，结果序列为GTGAGCAATCTGTCGCTCGATTTTTCTAA(SEQ ID No.8)。In this example, the vector pEcCas (addgene 73227) and pEcgRNA (addgene 166581) were used to knock out rarA in Escherichia coli stbl2 strain using CRISPR-Cas9. The editing site GGATAATACTTTTCAACCTC (SEQ ID No. 1) of rarA was selected, and the primers TAGTGGATAATACTTTT CAACCTC (SEQ ID No. 2) and AAACGAGGTTGAAAAGTATTATCC (SEQ ID No. 3) were annealed and inserted into the BsaI site of pEcgRNA to replace the fragment between the original two BsaI sites, thereby constructing the pEcgRNA A-rarA vector. Using primers CTTTGTCCTGACGCCGAAAGC (SEQ ID No.4) and CCGCA ACGATAACATTAGAAAAATCGAGCGACAGATTGC (SEQ ID No.5), the upstream homology arm of rar A was amplified by PCR, and the downstream homology arm of rarA was amplified by PCR using primers CGCTCGATTTTTCTAATGTTATCGTTGCGGTAATGTTGTT (SEQ ID No.6) and GGGAGTTACGCTCCGCTTGC (SEQ ID No.7). The two homology arms were spliced into the rarA knockout homology arm using overlapPCR. Gene knockout was performed according to the method described in the literature Q Li, B Sun, J Chen, et al. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin, 2021, 53(5), 620–627, deleting the DNA fragment at positions 27-1341 of rarA (1344bp) on the genome. The resulting sequence was GTGAGCAATCTGTCGCTCGATTTTTCTAA (SEQ ID No. 8).

敲除步骤如下：The knockout steps are as follows:

1)感受态制备：取含有pEcCas质粒的Stbl2单菌落接到5mL含卡那霉素的液体LB培养基中37℃过夜培养16h。按1％接种到10mL新鲜的含卡那霉素的LB培养基，37℃培养至OD600＝0.2。加入终浓度为10mM的阿拉伯糖后继续培养至OD600＝0.6。冰上放置15min后，冷冻离心机中3000rpm，4℃离心5min。10％预冷甘油重悬后离心，共洗涤3次。加入10％的预冷甘油使终体积为100uL。1) Preparation of competent state: Take a single colony of Stbl2 containing pEcCas plasmid and inoculate it into 5mL of liquid LB medium containing kanamycin and culture it at 37℃ overnight for 16h. Inoculate 1% into 10mL of fresh LB medium containing kanamycin and culture it at 37℃ until OD600=0.2. Add arabinose with a final concentration of 10mM and continue to culture until OD600=0.6. After placing on ice for 15min, centrifuge at 3000rpm and 4℃ in a refrigerated centrifuge for 5min. Resuspend in 10% pre-cooled glycerol and centrifuge, wash 3 times in total. Add 10% pre-cooled glycerol to make the final volume 100uL.

2)转化：取100ng pEcgRNA-rarA质粒，400ng rarA同源臂片段一起加入100μl Stbl2-pEcCas感受态。轻轻混合均匀后冰上放置3min，转入到预冷过的0.1mm电转杯中，电压设置1.8Kv，电击后立刻加入600μl LB，轻轻混匀后转入灭菌的1.5mL EP管中，37℃220rpm复苏45min，随后涂布含50μg/ml卡那霉素及50μg/ml壮观霉素的双抗平板，37℃过夜培养16h。2) Transformation: Take 100ng pEcgRNA-rarA plasmid and 400ng rarA homology arm fragment and add them to 100μl Stbl2-pEcCas competent medium. Mix gently and place on ice for 3 minutes, transfer to a pre-cooled 0.1mm electroporation cup, set the voltage to 1.8Kv, add 600μl LB immediately after electroporation, mix gently and transfer to a sterilized 1.5mL EP tube, resuscitate at 37℃220rpm for 45min, then apply double-resistance plate containing 50μg/ml kanamycin and 50μg/ml spectinomycin, and culture overnight at 37℃ for 16h.

3)PCR鉴定：随机挑取单菌落，利用引物CTTTGTCCTGACGCCGAAAGC(SEQ ID No.4)和GGGAGTTACGCTCCGCTTGC(SEQ ID No.7)进行菌落PCR验证。敲除成功的片段大小应为503bp，敲除失败的片段大小为1818bp。3) PCR identification: Randomly pick a single colony and perform colony PCR verification using primers CTTTGTCCTGACGCCGAAAGC (SEQ ID No. 4) and GGGAGTTACGCTCCGCTTGC (SEQ ID No. 7). The size of the fragment with successful knockout should be 503 bp, and the size of the fragment with failed knockout should be 1818 bp.

4)消除辅助质粒：将上述验证正确的单菌落接种于加了10mM鼠李糖和50μg/ml卡那霉素的3ml LB培养基，220rpm 37℃过夜培养16h。菌液用无菌水稀释十万倍后，取100μL涂布于含50μg/ml卡那霉素的LB平板，37℃过夜培养。生长起来的单菌落再用50μg/ml卡那霉素平板以及含50μg/ml卡那霉素和50μg/ml壮观霉素的平板筛选，对壮观霉素敏感的单菌落成功消除pEcgRNA。上述筛选得到的单菌落接种于3mL含有5g/L的葡萄糖的液体LB中，于37℃，220rpm过夜培养16h。然后将菌液涂布于含有5g/L葡萄糖及10g/l蔗糖的LB平板，37℃过夜培养16h。随机挑选单菌落于LB平板及含有50μg/ml卡那霉素的LB平板上，对卡那霉素敏感的菌落成功消除了pEcCas，甘油管保菌。4) Elimination of auxiliary plasmid: The single colony verified correctly above was inoculated into 3ml LB medium with 10mM rhamnose and 50μg/ml kanamycin, and cultured overnight at 220rpm and 37℃ for 16h. After the bacterial solution was diluted 100,000 times with sterile water, 100μL was spread on an LB plate containing 50μg/ml kanamycin and cultured overnight at 37℃. The grown single colonies were screened with 50μg/ml kanamycin plates and plates containing 50μg/ml kanamycin and 50μg/ml spectinomycin. The single colonies sensitive to spectinomycin successfully eliminated pEcgRNA. The single colonies obtained from the above screening were inoculated into 3mL liquid LB containing 5g/L glucose and cultured overnight at 37℃ and 220rpm for 16h. The bacterial solution was then spread on an LB plate containing 5g/L glucose and 10g/l sucrose, and cultured overnight at 37℃ for 16h. Single colonies were randomly selected and plated on LB plates and LB plates containing 50 μg/ml kanamycin. Colonies sensitive to kanamycin successfully eliminated pEcCas and glycerol-protected bacteria.

PCR鉴定rarA的敲除如图1所示，15号克隆成功敲除了目标片段。PCR identification of rarA knockout As shown in Figure 1, clone 15 successfully knocked out the target fragment.

将120个A的polyA(polyA120)插入pUC18质粒上，利用含有120个A(polyA120)的质粒验证polyA稳定性，将质粒转化后，利用PCR筛选polyA大小正确的克隆，将其接种到LB液体培养基中30℃培养20h后涂平板，随机挑选40个克隆，PCR扩增含polyA120的部分序列，利用琼脂糖凝胶电泳观察PCR产物的大小。A polyA of 120 A (polyA120) was inserted into the pUC18 plasmid, and the stability of polyA was verified using a plasmid containing 120 A (polyA120). After the plasmid was transformed, clones with the correct polyA size were screened by PCR. They were inoculated into LB liquid culture medium and cultured at 30°C for 20 hours, then spread on plates. 40 clones were randomly selected, and partial sequences containing polyA120 were amplified by PCR. The size of the PCR product was observed by agarose gel electrophoresis.

stbl2原始菌株的结果如图2所示，有约50％的克隆存在显著的polyA的缺失。The results of the stbl2 original strain are shown in FIG2 , and about 50% of the clones had a significant loss of polyA.

Stbl2-ΔrarA菌株约45％的克隆存在显著的polyA的缺失，从polyA基本完整的整体条带来看，rarA敲除菌株条带更单一清晰，敲除rarA对于polyA稳定性有一定的提升作用。About 45% of the clones of the Stbl2-ΔrarA strain showed a significant loss of polyA. Judging from the overall band of polyA that was basically intact, the band of the rarA knockout strain was more single and clear, and knocking out rarA had a certain effect on improving the stability of polyA.

实施例2、大肠杆菌stbl2中recE的敲除及其评价Example 2: Knockout of recE in Escherichia coli stbl2 and its evaluation

利用实施例1同样的方法，删除stbl2基因组上recE基因(2601bp)的47-288位的DNA片段，所删除片段的序列为：CCGGTGAACCTGACGTCGTCCTGTGGGCAAGCA ACGATTTTGAATCGACCTGTGCCACTCTGGACTACCTGATCGTTAAGTCAGGTAAAAAACTGAGCAGCTATTTTAAAGCTGTTGCCACGAATTTTCCTGTCGTTAATGACCTGCCCGCTGAAGGTGAGATCGATTTTACCTGGAGTGAACGCTATCAACTCAGCAAAGACTCCATGACATGGGAACTAAAACCGGGAGCAGCACCAGAC(SEQ ID No.9)。Using the same method as Example 1, the DNA fragment at positions 47-288 of the recE gene (2601bp) on the stbl2 genome was deleted. The sequence of the deleted fragment was: CCGGTGAACCTGACGTCGTCCTGTGGGCAAGCA ACGATTTTGAATCGACCTGTGCCACTCTGGACTACCTGATCGTTAAGTCAGGTAAAAAACTGAGCAGCTATTTTAAAGCTGTTGCCACGAATTTTCCTGTCGTTAATGACCTGCCCGCTGAAGGTGAGATCGATTTTACCTGGAGTGAACGCTATCAACTCAGCAAAGACTCCATGACATGGGAACTAAAACCGGGAGCAGCACCAGAC (SEQ ID No.9).

利用含polyA120的质粒验证stbl2-ΔrecE对polyA稳定性的影响，结果如图3所示，有62.5％的克隆存在显著的polyA缺失。可见敲除recE反而增加了polyA序列的不稳定性。The effect of stbl2-ΔrecE on polyA stability was verified using a plasmid containing polyA120. The results are shown in Figure 3. 62.5% of the clones had significant polyA deletions. It can be seen that knocking out recE actually increased the instability of the polyA sequence.

实施例3、大肠杆菌stbl2中敲除recF及其评价Example 3: Knockout of recF in Escherichia coli stbl2 and its evaluation

利用实施例1同样的方法，删除stbl2基因组上recF基因(1074bp)的347-621位的DNA片段，删除片段序列为：TGATAACGCCAGAAGGGTTTACTTTACTCAACGGCGGCCCCAAATACAGAAGAGCATTCCTCGACTGGGGATGCTTTCACAACGAACCCGGATTTTTCACCGCCTGGAGCAATCTCAAGCGATTGCTCAAGCAGCGCAATGCGGCGCTGCGCCAGGTGACACGTTACGAACAGCTACGCCCGTGGGATAAAGAGCTGATCCCGCTGGCGGAGCAAATCAGCACCTGGCGCGCGGAGTATAGCGCCGGTATCGCGGCCGATATGGCTGATACCTGT(SEQ ID No.10)。Using the same method as Example 1, the DNA fragment at positions 347-621 of the recF gene (1074 bp) on the stbl2 genome was deleted. The sequence of the deleted fragment was: TGATAACGCCAGAAGGGTTTACTTTACTCAACGGCGGCCCCAAATACAGAAGAGCATTCCTCGACTGGGGATGCTTTCACAACGAACCCGGATTTTTCACCGCCTGGAGCAATCTCAAGCGATTGCTCAAGCAGCGCAATGCGGCGCTGCGCCAGGTGACACGTTACGAACAGCTACGCCCGTGGGATAAAGAGCTGATCCCGCTGGCGGAGCAAATCAGCACCTGGCGCGCGGAGTATAGCGCCGGTATCGCGGCCGATATGGCTGATACCTGT (SEQ ID No.10).

利用含polyA120的质粒验证stbl2-ΔrecF对polyA稳定性的影响，结果如图4所示，仅有约17.5％的克隆存在明显的polyA序列的缺失。说明敲除recF可以显著提高polyA的稳定性。The effect of stbl2-ΔrecF on polyA stability was verified using a plasmid containing polyA120. The results are shown in Figure 4. Only about 17.5% of the clones had obvious polyA sequence deletions, indicating that knocking out recF can significantly improve the stability of polyA.

实施例4、大肠杆菌stbl2中敲除recJ及其评价Example 4: Knockout of recJ in Escherichia coli stbl2 and its evaluation

利用实施例1同样的方法，删除stbl2基因组上recJ基因(1734bp)的152-1076位的DNA片段，删除片段序列为GGCAGCAACTGAGCGGCGTCGAAAAGGCCGTTGAGATCCTTTACAACGCTTTTCGCGAAGGAACGCGGATTATTGTGGTCGGTGATTTCGACGCCGACGGCGCGACCAGCACGGCTCTAAGCGTGCTGGCGATGCGCTCGCTTGGTTGCAGCAATATCGACTACCTGGTACCAAACCGTTTCGAAGACGGTTACGGCTTAAGCCCGGAAGTGGTCGATCAGGCCCATGCCCGTGGCGCGCAGTTAATTGTCACGGTGGATAACGGTATTTCCTCCCATGCGGGGGTTGAGCACGCTCGCTCGTTGGGCATCCCGGTTATTGTTACCGATCACCATTTGCCAGGCGACACATTACCCGCAGCGGAAGCGATCATTAACCCTAACTTGCGCGACTGTAATTTCCCGTCGAAATCACTGGCAGGCGTGGGTGTGGCGTTTTATCTGATGCTGGCGCTGCGCACCTTTTTGCGCGATCAGGGCTGGTTTGATGAGCGTAACATCGCAATTCCTAACCTGGCAGAACTGCTGGATCTGGTCGCGCTGGGGACAGTGGCGGACGTCGTGCCGCTGGACGCTAATAATCGCATTCTGACCTGGCAGGGGATGAGTCGCATCCGAGCCGGAAAGTGCCGTCCGGGGATTAAAGCGCTGCTTGAAGTGGCAAACCGTGATGCACAAAAACTCGCCGCCAGCGATTTAGGTTTTGCGCTGGGGCCACGTCTCAATGCTGCCGGACGACTGGACGATATGTCCGTCGGTGTGGCGCTGTTGTTGTGCGACAACATCGGCGAAGCGCGCGTGCTGGCAAATGAACTCGATGCGCTAAACCAGACGCGAAAAGAGATCGAACAAGGAATGCAAATTGAAGCCCTGACCCTGTGCGAGAAACTGGAGCGCAGCCGTGACACGCTACCCGGCGGGCTGGC(SEQ ID No.11)。Using the same method as in Example 1, the DNA fragment at positions 152-1076 of the recJ gene (1734 bp) on the stbl2 genome was deleted, and the sequence of the deleted fragment was GGCAGCAACTGAGCGGCGTCGAAAAGGCCGTTGAGATCCTTTACAACGCTTTTCGCGAAGGAACGCGGATTATTGTGGTCGGTGATTTCGACGCCGACGGCGCGACCAGCACGGCTCTAAGCGTGCTGGCGATGCGCTCGCTTGGTTGCAGCAATATCGACTACCTGGTACCAAACCGTTTCGAAGACG GTTACGGCTTAAGCCCGGAAGTGGTCGATCAGGCCCATGCCCGTGGCGCGCAGTTAATTGTCACGGTGGATAACGGTATTTCCTCCCATGCGGGGGTTGAGCACGCTCGCTCGTTGGGCATCCCGGTTATTGTTACCGATCACCATTTGCCAGGCGACACATTACCCGCAGCGGAAGCGATCATTAACCCTAACTTGCGCGACTGTAATTTCCCGTCGAAATCACTGGCAGGCGTGGGTGTGGCGTTTTA TCTGATGCTGGCGCTGCGCACCTTTTTTGCGCGATCAGGGCTGGTTTGATGAGCGTAACATCGCAATTCCTAACCTGGCAGAACTGCTGGATCTGGTCGCGCTGGGGACAGTGGCGGACGTCGTGC CGCTGGACGCTAATAATCGCATTCTGACCTGGCAGGGGATGAGTCGCATCCGAGCCGGAAAGTGCCGTCCGGGGATTAAAGCGCTGCTTGAAGTGGCAAACCGTGATGCACAAAAACTCGCCGCC AGCGATTTAGGTTTTGCGCTGGGGCCACGTCTCAATGCTGCCGGACGACTGGACGATATGTCCGTCGGTGTGGCGCTGTTGTTGTGCGACAACATCGGCGAAGCGCGCGTGCTGGCAAATGAACT CGATGCGCTAAACCAGACGCGAAAAGAGATCGAACAAGGAATGCAAATTGAAGCCCTGACCCTGTGCGAGAAACTGGAGCGCAGCCGTGACACGCTACCCGGCGGGCTGGC (SEQ ID No. 11).

利用含polyA120的质粒验证stbl2-ΔrecJ对polyA稳定性的影响，结果约25％的克隆存在明显的polyA缺失。说明敲除recJ可以显著提高polyA的稳定性。The effect of stbl2-ΔrecJ on polyA stability was verified using a plasmid containing polyA120, and the results showed that about 25% of the clones had obvious polyA deletions, indicating that knocking out recJ can significantly improve the stability of polyA.

实施例5、大肠杆菌stbl2中敲除recO或recR及其评价Example 5: Knockout of recO or recR in Escherichia coli stbl2 and its evaluation

利用实施例1同样的方法，分别删除stbl2基因组上recO基因(729bp)的第1-729位的DNA片段和stbl2基因组上recR(606bp)基因的1-603位的DNA片段。其中，recO删除片段序列为ATGGAAGGCTGGCAGCGCGCATTTGTCCTGCATAGTCGCCCGTGGAGCGAAACCAGCCTGATGCTGGACGTCTTCACGGAGGAATCGGGGCGCGTGCGTCTGGTTGCCAAAGGCGCACGCTCTAAACGCTCTACCCTGAAAGGTGCATTACAGCCTTTCACCCCTCTCTTGCTACGTTTTGGCGGGCGTGGCGAAGTCAAAACGCTGCGCAGTGCTGAAGCCGTCTCGCTGGCGCTGCCATTAAGCGGTATCACGCTTTACAGCGGTCTGTACATCAACGAACTTCTCTCCCGCGTACTGGAATACGAGACGCGCTTCTCTGAACTTTTTTTCGATTACTTGCACTGCATTCAGTCTCTTGCAGGGGTCACTGGTACGCCAGAACCCGCGCTGCGCCGCTTTGAACTGGCACTGCTCGGGCATCTGGGTTATGGCGTCAATTTTACCCATTGTGCGGGTAGCGGCGAGCCGGTAGATGACACCATGACGTATCGTTATCGCGAAGAAAAAGGGTTTATCGCAAGCGTCGTTATCGACAATAAAACGTTCACCGGAAGGCAGTTAAAAGCGTTAAACGCACGGGAATTTCCTGACGCAGACACACTGCGCGCCGCGAAACGCTTTACCCGCATGGCGCTTAAGCCGTATCTTGGCGGTAAACCTTTAAAGAGCAGGGAACTGTTCCGGCAGTTTATGCCTAAGCGAACGGTGAAAACACATTATGAATGA(SEQ ID No.12)。RecR删除片段序列为：ATGCAAACCAGCCCGCTGTTAACACAGCTTATGGAAGCACTGCGCTGTCTGCCGGGCGTTGGCCCGAAGTCGGCGCAGCGTATGGCGTTCACGCTGCTTCAGCGCGATCGTAGCGGCGGGATGCGTCTGGCGCAGGCGCTCACCCGGGCGATGTCGGAAATCGGCCACTGCGCCGATTGCCGCACTTTCACCGAACAGGAAGTCTGTAACATCTGTTCGAATCCGCGTCGTCAGGAAAACGGTCAAATCTGCGTGGTGGAGAGTCCGGCGGACATCTACGCCATTGAGCAGACGGGGCAGTTTTCAGGTCGTTATTTTGTGTTGATGGGGCATCTGTCACCGCTGGACGGCATCGGTCCGGATGATATCGGGCTTGATCGTCTGGAACAGCGTCTGGCAGAGGAAAAAATCACTGAAGTGATCCTCGCCACCAACCCCACGGTTGAAGGTGAAGCTACCGCTAACTACATTGCCGAGCTTTGCGCGCAATATGACGTGGAAGCCAGCCGAATCGCTCATGGCGTTCCGGTTGGCGGCGAGCTGGAAATGGTCGACGGCACCACGTTGTCACACTCCCTTGCCGGGCGTCATAAGATTCGTTTT(SEQ ID No.13)。在本实施例中，随机挑选了72个克隆进行PCR验证。Using the same method as in Example 1, the DNA fragment at positions 1-729 of the recO gene (729 bp) on the stbl2 genome and the DNA fragment at positions 1-603 of the recR gene (606 bp) on the stbl2 genome were deleted respectively. Among them, the recO deletion fragment sequence is ATGGAAGGCTGGCAGCGCGCATTTGTCCTGCATAGTCGCCCGTGGAGCGAAACCAGCCTGATGCTGGACGTCTTCACGGAGGAATCGGGGCGCGTGCGTCTGGTTGCCAAAGGCGCACGCTCTAAACGCTCTACCCTGAAAGGTGCATTACAGCCTTTCACCCCTCTCTTGCTAC GTTTTGGCGGGCGTGGCGAAGTCAAAACGCTGCGCAGTGCTGAAGCCGTCTCGCTGGCGCTGCCATTAAGCGGTATCACGCTTTACAGCGGTCTGTACATCAACGAACTTCTCTCCCGCGTACTGGAATACGAGACGCGCTTCTCTGAACTTTTTTCGATTACTTGCACTGCATTCAGTCTCTTGCAGG GGTCACTGGTACGCCAGAACCCGCGCTGCGCCGCTTTGAACTGGCACTGCTCGGGCATCTGGGTTATGGCGTCAATTTTACCCATTGTGCGGGTAGCGGCGAGCCGGTAGATGACACCATGACGTATCGTTATCGCGAAGAAAAAGGGTTTATCGCAAGCGTCGTTATCGACAATAAAACGTTCACCGG AAGGCAGTTAAAAGCGTTAAACGCACGGGAATTTCCTGACGCAGACACACTGCGCGCCGCGAAACGCTTTACCCGCATGGCGCTTAAGCCGTATCTTGGCGGTAAAACCTTTAAAGAGCAGGGAACTGTTCCGGCAGTTTATGCCTAAGCGAACGGTGAAAACACATTATGAATGA (SEQ ID No. 12). The sequence of the RecR deletion fragment is: ATGCAAACCAGCCCGCTGTTAACACAGCTTATGGAAGCACTGCGCTGTCTGCCGGGCGTTGGCCCGAAGTCGGCGCAGCGTATGGCGTTCACGCTGCTTCAGCGCGATCGTAGCGGCGGGATGCGTCTGGCGCAGGCGCTCACCC GGGCGATGTCGGAAATCGGCCACTGCGCCGATTGCCGCACTTTCACCGAACAGGAAGTCTGTAACATCTGTTCGAATCCGCGTCGTCAGGAAAACGGTCAAATCTGCGTGGTGGAGAGTCCGGCGGACATCTACGCCATTGAGCAGACGGGGCAGTTT TCAGGTCGTTATTTTGTGTTGATGGGGCATCTGTCACCGCTGGACGGCATCGGTCCGGATGATATCGGGCTTGATCGTCTGGAACAGCGTCTGGCAGAGGAAAAAATCACTGAAGTGATCCTCGCCACCAACCCCACGGTTGAAGGTGAAGCTACCG CTAACTACATTGCCGAGCTTTGCGCGCAATATGACGTGGAAGCCAGCCGAATCGCTCATGGCGTTCCGGTTGGCGGCGAGCTGGAAATGGTCGACGGCACCACGTTGTCACACTCCCTTGCCGGGCGTCATAAGATTCGTTTT (SEQ ID No. 13). In this example, 72 clones were randomly selected for PCR verification.

利用含polyA120的质粒分别验证stbl2-ΔrecO和stbl2-ΔrecR对polyA稳定性的影响，结果存在明显polyA缺失的克隆的比例分别为52.8％(38/72)和47.2％(34/72)，和stbl2原始菌株接近。可见敲除recO或recR并不影响polyA的稳定性。Plasmids containing polyA120 were used to verify the effects of stbl2-ΔrecO and stbl2-ΔrecR on polyA stability. The results showed that the proportions of clones with obvious polyA deletion were 52.8% (38/72) and 47.2% (34/72), respectively, which were close to the original strain of stbl2. It can be seen that knocking out recO or recR does not affect the stability of polyA.

实施例6、大肠杆菌DH5α中敲除recF和/或recJ及其评价Example 6: Knockout of recF and/or recJ in Escherichia coli DH5α and evaluation thereof

在DH5α菌株中利用实施例1同样的方法分别敲除recF和recJ，利用polyA120评价其稳定性，结果DH5α、DH5α-ΔrecF、DH5α-ΔrecJ菌株中存在明显polyA缺失的比例分别为72.2％，52.1％和68.1％，可见敲除recF和recJ有助于提高polyA的稳定性，尤其是敲除recF可大幅提高polyA120的稳定性，和在stbl2菌株中进行敲除的结果一致。In the DH5α strain, recF and recJ were knocked out respectively using the same method as in Example 1, and their stability was evaluated using polyA120. The results showed that the proportions of obvious polyA deletions in the DH5α, DH5α-ΔrecF, and DH5α-ΔrecJ strains were 72.2%, 52.1%, and 68.1%, respectively. It can be seen that knocking out recF and recJ helps to improve the stability of polyA, especially knocking out recF can greatly improve the stability of polyA120, which is consistent with the results of knocking out in the stbl2 strain.

进一步将recF和recJ同时敲除，菌株DH5α-ΔrecFΔrecJ中存在明显polyA120缺失的比例为47.5％。说明在DH5α中，同时敲除recF和recJ相对单独敲除，进一步提高了polyA120的稳定性。Further, when recF and recJ were knocked out simultaneously, the proportion of obvious polyA120 deletion in the strain DH5α-ΔrecFΔrecJ was 47.5%, indicating that in DH5α, knocking out recF and recJ simultaneously further improved the stability of polyA120 compared with knocking out each alone.

实施例7、在stbl2-ΔrecF的基础上敲除recJ及其性能鉴定Example 7: Knockout of recJ based on stbl2-ΔrecF and performance evaluation thereof

采用实施例1同样的方法，在stbl2-ΔrecF的基础上敲除recJ，构建菌株stbl2-ΔrecFΔrecJ。The same method as in Example 1 was used to knock out recJ on the basis of stbl2-ΔrecF to construct the strain stbl2-ΔrecFΔrecJ.

利用polyA120评价其稳定性，结果stbl2-ΔrecFΔrecJ中存在明显polyA缺失的比例为36.1％。说明同时敲除recF和recJ也有助于提高polyA120的稳定性。The stability of polyA120 was evaluated, and the results showed that the proportion of obvious polyA deletion in stbl2-ΔrecFΔrecJ was 36.1%, indicating that knocking out recF and recJ at the same time can also help improve the stability of polyA120.

实施例8、在stbl2-ΔrecE的基础上敲除recF或进一步敲除recJ及其性能鉴定Example 8: Knockout of recF or further knockout of recJ based on stbl2-ΔrecE and performance evaluation thereof

采用实施例1同样的方法，在stbl2-ΔrecE的基础上敲除recF，构建菌株stbl2-ΔrecEΔrecF，然后在此菌株的基础上进一步敲除recJ，构建菌株stbl2-ΔrecEΔrecFΔrecJ。The same method as in Example 1 was used to knock out recF on the basis of stbl2-ΔrecE to construct the strain stbl2-ΔrecEΔrecF, and then on the basis of this strain, recJ was further knocked out to construct the strain stbl2-ΔrecEΔrecFΔrecJ.

利用polyA120评价其稳定性，结果stbl2-ΔrecEΔrecF中存在明显polyA缺失的比例为12.5％，stbl2-ΔrecEΔrecFΔrecJ中存在明显polyA缺失的比例仅为11.6％(如图5所示)。而stbl2-ΔrecE中存在明显polyA缺失的比例为62.5％。进一步证明recF和recJ的敲除有助于提高polyA120的稳定性。The stability was evaluated by polyA120, and the results showed that the proportion of stbl2-ΔrecEΔrecF with obvious polyA deletion was 12.5%, and the proportion of stbl2-ΔrecEΔrecFΔrecJ with obvious polyA deletion was only 11.6% (as shown in Figure 5). The proportion of stbl2-ΔrecE with obvious polyA deletion was 62.5%, which further proved that the knockout of recF and recJ helped to improve the stability of polyA120.

实施例9、在stbl2-ΔrecEΔrecF的基础上敲除rarA及其性能鉴定Example 9: Knockout of rarA based on stbl2-ΔrecEΔrecF and performance evaluation thereof

采用实施例1同样的方法，在stbl2-ΔrecEΔrecF的基础上敲除rarA，构建菌株stbl2-ΔrecEΔrecFΔrarA，利用polyA120评价菌株对polyA稳定性的影响，结果存在明显polyA缺失的比例为55％，高于stbl2-ΔrecEΔrecF。敲除rarA并不能进一步提高stbl2-ΔrecEΔrecF的polyA120的稳定性。The same method as in Example 1 was used to knock out rarA on the basis of stbl2-ΔrecEΔrecF to construct the strain stbl2-ΔrecEΔrecFΔrarA, and the effect of the strain on polyA stability was evaluated using polyA120. The results showed that the proportion of obvious polyA deletion was 55%, which was higher than that of stbl2-ΔrecEΔrecF. Knocking out rarA did not further improve the stability of polyA120 of stbl2-ΔrecEΔrecF.

实施例10、在stbl2-ΔrecEΔrecF的基础上敲除DNA聚合酶及其评价Example 10: Knockout of DNA polymerase based on stbl2-ΔrecEΔrecF and its evaluation

采用实施例1同样的方法，在stbl2-ΔrecEΔrecF的基础上分别以及同时敲除易错的DNA聚合酶pol II(polB，2352bp)和DNA聚合酶pol V(1269bp)，其中pol II删除17-747位的DNA片段，其序列为：TTATCTTAACCCGACACTGGCGGGACACCCCGCAAGGGACAGAAGTCTCCTTCTGGCTGGCGACGGACAACGGGCCGTTGCAGGTTACGCTTGCACCGCAAGAGTCCGTGGCGTTTATTCCCGCCGATCAGGTTCCCCGCGCTCAGCATATTTTGCAGGGTGAACAAGGCTTTCGCCTGACACCGCTGGCGTTAAAGGATTTTCACCGCCAGCCGGTGTATGGCCTTTACTGTCGCGCCCATCGCCAATTGATGAATTACGAAAAGCGCCTGCGTGAAGGTGGCGTTACCGTCTACGAGGCCGATGTGCGTCCGCCAGAACGCTATCTGATGGAGCGGTTTATCACCTCACCGGTGTGGGTCGAGGGTGATATGCACAATGGCACTATCGTTAATGCCCGTCTGAAACCGCATCCCGACTATCGTCCGCCGCTCAAGTGGGTTTCTATAGATATTGAAACCACCCGCCACGGTGAGCTGTACTGCATCGGCCTGGAAGGCTGCGGGCAGCGCATCGTTTATATGCTGGGGCCGGAGAATGGCGACGCCTCCTCGCTTGATTTCGAACTGGAATACGTCGCCAGCCGCCCGCAGTTGCTGGAAAAACTCAACGCCTGGTTTGCCAACTACGATCCTGATGTGATCATCGGTTGGAACGTGGTGCAGTTCGATCTGCGAATGCTGCAAAAACATGCCGAGCGTTACCGTCTTCCGCTGCGTCTTGGGCGCGAT(SEQ ID No.14)，pol V删除370-1223位的DNA片段，其序列为：CGCGCAACGGTGCTACAACGTACCCATCTTACTGTTGGTGTGGGGATCGCCCAGACCAAAACGCTGGCTAAGCTTGCCAATCATGCGGCAAAAAAATGGCAGCGGCAGACGGGTGGGGTGGTGGATTTATCAAATCTGGAACGCCAGCGTAAATTAATGTCTGCTCTCCCCGTGGATGACGTCTGGGGGATTGGACGGCGGATCAGCAAAAAACTGGACGCGATGGGGATCAAAACCGTTCTCGATTTGGCGGATACAGATATCCGGTTTATCCGTAAACATTTTAATGTCGTGCTCGAAAGAACGGTGCGTGAACTGCGCGGCGAACCCTGTTTGCAACTGGAAGAGTTTGCACCGACGAAGCAGGAAATTATCTGTTCCCGCTCGTTTGGTGAACGCATCACGGATTATCCGTCGATGCGGCAGGCCATTTGTAGTTACGCTGCCCGGGCGGCGGAAAAACTTCGCAGCGAGCATCAATATTGTCGGTTTATCTCCACGTTTATTAAGACGTCACCATTTGCGCTCAATGAACCTTATTACGGCAATAGCGCGTCGGTAAAACTGCTGACGCCCACTCAGGACAGCAGGGATATCATTAACGCTGCTACGCGATCTCTGGATGCCATCTGGCAAGCGGGCCATCGTTACCAAAAAGCGGGCGTGATGCTGGGGGATTTCTTCAGTCAGGGAGTCGCGCAGCTCAATTTATTCGATGACAACGCACCGCGCCCCGGGAGTGAGCAATTGATGACGGTAATGGATACACTGAATGCTAAAGAGGGCAGAGGAACACTCTATTTTGCCGGGCAGGGGATCCAGCAACAATGGCAGATGAAGCGAGCCATGCTTTC(SEQ ID No.15)。The same method as in Example 1 was used to knock out the error-prone DNA polymerase pol II (polB, 2352 bp) and DNA polymerase pol V (1269 bp) separately and simultaneously on the basis of stbl2-ΔrecEΔrecF, wherein pol II deleted the DNA fragment at positions 17-747, whose sequence was: TTATCTTAACCCGACACTGGCGGGACACCCCGCAAGGGACAGAAGTCTCCTTCTGGCTGGCGACGGACAACGGGCCGTTGCAGGTTACGCTTGCACCGCAAGAGTCCGTGGCGTTTATTCCCGCCGATCAGGTTCCCCGCGCTCAGCATATTTTGCAGGGTGAACAAGGCTTTCGCCTGACACCGCTGGCGTTAAAGGATTTTCACCGCCAGCCGGTGTATGGCCTTTACTGTCGCGCCCATCGCCAATTGATGAATTACGAAAAGCGCCTGCGTGAAGGTGGCGTTACCGTCTACGAGGCCGATGTGCGTCCGCCAGAAC GCTATCTGATGGAGCGGTTTATCACCTCACCGGTGTGGGTCGAGGGTGATATGCACAATGGCACTATCGTTAATGCCCGTCTGAAACCGCATCCCGACTATCGTCCGCCGCTCAAGTGGGTTTCTATAGATATTGAAACCACCCGCCACGGTGAGCTGTACTGCATCGGCCTGGAAGGCTGCGGGCAGCGCATCGTTTATATGCTGGGGCCGGAGAATGG CGACGCCTCCTCGCTTGATTTCGAACTGGAATACGTCGCCAGCCGCCCGCAGTTGCTGGAAAAACTCAACGCCTGGTTTGCCAACTACGATCCTGATGTGATCATCGGTTGGAACGTGGTGCAGTTCGATCTGCGAATGCTGCAAAAACATGCCGAGCGTTACCGTCTTCCGCTGCGTCTTGGGCGCGAT (SEQ ID No.14), pol V deletes bits 370-1223 The DNA fragment has the sequence: CGCGCAACGGTGCTACAACGTACCCATCTTACTGTTGGTGTGGGGATCGCCCAGACCAAAACGCTGGCTAAGCTTGCCAATCATGCGGCAAAAAAATGGCAGCGGCAGACGGGTGGTGGTGGATTTATCAAATCTGGAACGCCAGCGTAAATTAATGTCTGCTCTCCCCGTGGATGACGTCTGGGGGATTGGACGGCGGATCAGCA AAAAACTGGACGCGATGGGGATCAAAACCGTTCTCGATTTGGCGGATACAGATATCCGGTTTATCCGTAAACATTTTAATGTCGTGCTCGAAAGAACGGTGCGTGAACTGCGCGGCGAACCCTGTTTGCAACTGGAAGAGTTTGCACCGACGAAGCAGGAAATTATCTGTTCCCGCTCGTTTGGTGAACGCATCACGGATTATCCGTCGATGCGGCAGGC CATTTGTAGTTACGCTGCCCGGGCGGCGGAAAAACTTCGCAGCGAGCATCAATATTGTCGGTTTATCTCCACGTTTATTAAGACGTCACCATTTGCGCTCAATGAACCTTATTACGGCAATAGCGCGTCGGTAAAACTGCTGACGCCCACTCAGGACAGCAGGGATATCATTAACGCTGCTACGCGATCTCTGGATGCCATCTGGCAAGCGGGCCATCGT TACCAAAAAGCGGGCGTGATGCTGGGGGATTTCTTCAGTCAGGGAGTCGCGCAGCTCAATTTATTCGATGACAACGCACCGCGCCCCGGGAGTGAGCAATTGATGACGGTAATGGATACACTGAATGCTAAAGAGGGCAGAGGAACACTCTATTTTGCCGGGCAGGGGATCCAGCAACAATGGCAGATGAAGCGAGCCATGCTTTC (SEQ ID No. 15).

构建得到3个菌株：stbl2-ΔrecEΔrecFΔpol II、stbl2-ΔrecEΔrecFΔpol V和stbl2-ΔrecEΔrecFΔpol IIΔpol V。Three strains were constructed: stbl2-ΔrecEΔrecFΔpol II, stbl2-ΔrecEΔrecFΔpol V and stbl2-ΔrecEΔrecFΔpol IIΔpol V.

同样利用polyA120评价菌株对polyA稳定性的影响，结果这3个菌株存在明显polyA缺失的比例分别为52.5％、30％和60％，均高于stbl2-ΔrecEΔrecF的12.5％。说明敲除易错的DNA聚合酶并不能有效提高polyA的稳定性。PolyA120 was also used to evaluate the effect of strains on polyA stability. The results showed that the proportions of obvious polyA deletion in these three strains were 52.5%, 30% and 60%, respectively, which were all higher than 12.5% of stbl2-ΔrecEΔrecF. This shows that knocking out error-prone DNA polymerases cannot effectively improve the stability of polyA.

表1利用polyA120评价菌株

Table 1 Evaluation of strains using polyA120

实施例11、利用polyA90验证stbl2-ΔrecF和stbl2-ΔrecEΔrecF中polyA的稳定性Example 11: Verification of the stability of polyA in stbl2-ΔrecF and stbl2-ΔrecEΔrecF using polyA90

将含有90个连续A(polyA90)的质粒分别导入stbl2-ΔrecF和stbl2-ΔrecEΔrecF中，30℃培养20h后涂布LB平板，挑选72个单克隆PCR扩增含polyA90的片段，琼脂糖凝胶电泳鉴定条带大小，stbl2、stbl2-ΔrecF和stbl2-ΔrecEΔrecF中，存在明显polyA缺失的比例分别为2.8％、4.2％和2.8％。由于polyA90的串联重复序列较短，在30℃培养条件下均较稳定，不同宿主菌之间无明显差异。Plasmids containing 90 consecutive A (polyA90) were introduced into stbl2-ΔrecF and stbl2-ΔrecEΔrecF, respectively. After culturing at 30°C for 20 hours, LB plates were coated and 72 monoclonal PCR fragments containing polyA90 were selected. The band size was identified by agarose gel electrophoresis. The proportions of stbl2, stbl2-ΔrecF and stbl2-ΔrecEΔrecF with obvious polyA deletion were 2.8%, 4.2% and 2.8%, respectively. Since the tandem repeat sequence of polyA90 is short, it is relatively stable under 30°C culture conditions, and there is no significant difference between different host bacteria.

将含有90个连续A(polyA90)的质粒分别导入stbl2-ΔrecF和stbl2-ΔrecEΔrecF中，37℃培养20h后涂布LB平板，随机挑选克隆PCR扩增含polyA90的片段，琼脂糖凝胶电泳鉴定条带大小，stbl2、stbl2-ΔrecF和stbl2-ΔrecEΔrecF中，存在明显polyA缺失的比例分别为94.4％、91.7％和58.3％。Plasmids containing 90 consecutive A's (polyA90) were introduced into stbl2-ΔrecF and stbl2-ΔrecEΔrecF, respectively. After culture at 37°C for 20 h, the plates were spread on LB plates. Clones were randomly selected and PCR amplified for fragments containing polyA90. The band sizes were identified by agarose gel electrophoresis. The proportions of stbl2, stbl2-ΔrecF and stbl2-ΔrecEΔrecF with obvious polyA deletion were 94.4%, 91.7% and 58.3%, respectively.

实施例12、利用多段化polyA验证stbl2、stbl2-ΔrecF、stbl2-ΔrecEΔrecF中polyA的稳定性Example 12: Verification of the stability of polyA in stbl2, stbl2-ΔrecF, and stbl2-ΔrecEΔrecF using multi-segmented polyA

将含有多段化polyA的质粒(多段化polyA的序列为：AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA，SEQ ID No.16)分别转化stbl2、stbl2-ΔrecF、stbl2-ΔrecEΔrecF菌株，液体LB培养基中30℃培养24h后，转接到新的LB培养基中，连续转接培养10次，涂布LB固体平板，挑96个单克隆进行PCR验证polyA的缺失率，结果stbl2、stbl2-ΔrecF、stbl2-ΔrecEΔrecF中，存在明显缺失的克隆比例分别为21.9％、6.3％和18.9％。The plasmid containing multi-segmented polyA (the sequence of multi-segmented polyA is: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA, SEQ ID No.16) was transformed into stbl2, stbl2-ΔrecF, and stbl2-ΔrecEΔrecF strains, respectively. After culturing in liquid LB medium at 30°C for 24 hours, the cells were transferred to new LB medium and transferred and cultured for 10 times. The cells were spread on LB solid plates, and 96 single clones were selected for PCR verification of the polyA deletion rate. The results showed that the proportions of clones with obvious deletions in stbl2, stbl2-ΔrecF, and stbl2-ΔrecEΔrecF were 21.9%, 6.3%, and 18.9%, respectively.

以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

一种工程化大肠杆菌菌株，其recF和/或recJ蛋白低表达或不表达，或者表达缺失或无活性的recF和/或recJ蛋白。An engineered Escherichia coli strain, wherein the recF and/or recJ proteins are lowly expressed or not expressed, or the recF and/or recJ proteins are deleted or inactive. 根据权利要求1所述的工程化大肠杆菌菌株，其基因组中的recF和/或recJ的编码序列被全部或部分地截短或者被突变，使得所述菌株recF和/或recJ蛋白低表达或不表达，或者表达缺失或无活性的recF和/或recJ蛋白。According to the engineered Escherichia coli strain of claim 1, the coding sequence of recF and/or recJ in its genome is completely or partially truncated or mutated, so that the strain recF and/or recJ protein is low-expressed or not expressed, or expresses missing or inactive recF and/or recJ protein. 根据权利要求1或2所述的工程化大肠杆菌菌株，其recE蛋白低表达或不表达，或者表达缺失或无活性的recE蛋白。The engineered Escherichia coli strain according to claim 1 or 2, wherein the recE protein is lowly expressed or not expressed, or the recE protein is missing or inactive. 根据权利要求1-3任一项所述的工程化大肠杆菌菌株，其DNA聚合酶V低表达或不表达，或者表达缺失或无活性的DNA聚合酶V；The engineered Escherichia coli strain according to any one of claims 1 to 3, wherein the DNA polymerase V is lowly expressed or not expressed, or the DNA polymerase V is missing or inactive; 优选地，所述工程化大肠杆菌菌株的DNA聚合酶II无缺陷表达。Preferably, the engineered E. coli strain has no defective expression of DNA polymerase II. 根据权利要求1-4任一项所述的工程化大肠杆菌菌株，其为工程化的DH5α或stbl2菌株。The engineered Escherichia coli strain according to any one of claims 1 to 4, which is an engineered DH5α or stbl2 strain. 权利要求1-5任一项所述的工程化大肠杆菌菌株的制备方法，其包括：The method for preparing the engineered Escherichia coli strain according to any one of claims 1 to 5, comprising: 采用点突变、删除部分或整个ORF阅读框的方法，使得大肠杆菌菌株的所述蛋白低表达或不表达，或者表达缺失或无活性的所述蛋白。By using point mutation or deleting part or the whole ORF reading frame, the protein of the E. coli strain is lowly expressed or not expressed, or the protein is missing or inactive. 权利要求1-5任一项所述的工程化大肠杆菌菌株在生产质粒中的应用；Use of the engineered Escherichia coli strain according to any one of claims 1 to 5 in producing plasmids; 优选地，所述质粒为含有串联重复序列例如polyA的质粒。Preferably, the plasmid is a plasmid containing a tandem repeat sequence such as polyA. 一种制备含有polyA序列的质粒的方法，该方法包括：A method for preparing a plasmid containing a polyA sequence, the method comprising: 采用权利要求1-5任一项所述的工程化大肠杆菌菌株发酵制备含有polyA序列的质粒。The engineered Escherichia coli strain according to any one of claims 1 to 5 is used to ferment and prepare a plasmid containing a polyA sequence. 根据权利要求8所述的方法，其中，所述发酵条件为：28-32℃培养12h-72h。The method according to claim 8, wherein the fermentation conditions are: culturing at 28-32°C for 12h-72h. 根据权利要求8或9所述的方法，其中，所述polyA序列长度为24nt-144nt优选为90nt-120nt。The method according to claim 8 or 9, wherein the polyA sequence length is 24nt-144nt, preferably 90nt-120nt.
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