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Rhodococcus gene editing method using phenylalanyl-tRNA synthetase gene mutant as reverse selection marker

A phenylalanyl and gene editing technology, applied in the field of gene editing, can solve the problems of antibiotic resistance gene leakage, affecting cell traits, etc., and achieve the effect of efficient screening and high specificity

Active Publication Date: 2022-06-24
NANJING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The residues of exogenous nucleotide fragments may not only affect the traits of cells, but also the residues of antibiotic markers may cause the leakage of antibiotic resistance genes, resulting in certain biological safety (The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630[J].Applied microbiology and biotechnology, 2013, 97(5): 2119-2130.)

Method used

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  • Rhodococcus gene editing method using phenylalanyl-tRNA synthetase gene mutant as reverse selection marker
  • Rhodococcus gene editing method using phenylalanyl-tRNA synthetase gene mutant as reverse selection marker
  • Rhodococcus gene editing method using phenylalanyl-tRNA synthetase gene mutant as reverse selection marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Rhodococcus opacus Deletion of the mucofuroate cycloisomerase gene (GenBank:AHK33752.1) in PD630

[0030] (1) Construction of pk18mob-pheS plasmid

[0031] extract R. opacus PD630 genome, and design specific primers to clone the nucleotide fragment containing phenylalanyl-tRNA synthetase gene (AHK32253.1) and its own promoter, wherein the upstream primer is SEQ ID NO: 1: tacccggggatcctctagaTGCGGTCCTCGACAGCATCAGCG; downstream The primer is SEQ ID NO: 2: aacgacggccagtgccaagcttATTGCGCTACTCGCACGTCTGC, and the PCR reaction is carried out according to the following system:

[0032] 5×Reaction buffer 10.0 μL dNTPs (10 mM) 1.0 μL Upstream primer SEQ ID NO: 1 (10 μM) 1.0 μL Downstream primer SEQ ID NO: 2 (10 μM) 1.0 μL Rhodococcus opacus PD630 Genome

1.0 μL Taq enzyme (5 U / μL) 1.0 μL ddH 2 O

35.0 μL Overall system 50 μL

[0033] The reaction system was performed as follows: pre-denaturat...

Embodiment 2

[0046] Example 2 Rhodococcus opacus Deletion of the catechol 2,3 dioxygenase gene in PD630 (GenBank: AHK27425.1)

[0047] (1) The construction of pk18mob-pheS* plasmid is the same as steps (1) and (2) in Example 1.

[0048] (2) Insert the nucleotide fragments on both sides of the catechol 2,3 dioxygenase gene locus into the pk18mob-pheS* anti-screening plasmid

[0049] 通过设计特异性引物:SEQ ID NO:11:acgaattcgagctcggtaccGGCCAACGGCGTGAAGCCGGC;SEQ ID NO:12:caggcccccacaccgaggacaactcGACACGGGACGCACCGTCGAAAGGGAC;SEQID NO:13:gtccctttcgacggtgcgtcccgtgtcGAGTTGTCCTCGGTGTGGGGGCCTG;SEQ ID NO:14:tgtcgaggaccgcatctagaGAGCGGGACGACCTCCTGCTGCG,通过PCR及上述引物获得邻苯二酚2,3 A nucleotide fragment of approximately 1000 bp upstream and downstream of the dioxygenase gene. The nucleotide fragments on both sides were ligated together by overlapping PCR method, and then the ligated nucleotide fragments were inserted into the pk18mob-pheS* plasmid using an assembly kit to obtain a suicide plasmid containing homology a...

Embodiment 3

[0054] Example 3 Rhodococcus opacus Deletion of the protocatechuate 3,4 dioxygenase gene in PD630 (GenBank: AHK32653.1)

[0055] (1) The construction of pk18mob-pheS* plasmid is the same as steps (1) and (2) in Example 1.

[0056] (2) Insert the nucleotide fragments on both sides of the protocatechuate 3,4 dioxygenase gene locus into the pk18mob-pheS* counter-screening plasmid

[0057] 通过设计特异性引物:SEQ ID NO:17:acgaattcgagctcggtaccCGGCCCGACCCCGAGGATGC;SEQ ID NO:18:gcgcgacacctttctgggttgtgaccgaGGAAAAAGATCCTCACGTTCTCGATGTGAACAGTC;SEQ ID NO:19:gactgttcacatcgagaacgtgaggatctttttccTCGGTCACAACCCAGAAAGGTGTCGCGC;SEQ ID NO:20:gcctgcaggtcgactctagaGCGCGACGGCTCCGCCGG,通过PCR及上述引物获得原儿茶酸3, 4 Nucleotide fragments of about 1000 bp upstream and downstream of the dioxygenase gene. The upstream and downstream fragments of the protocatechuate 3,4 dioxygenase gene were ligated together by overlapping PCR method, and then the ligated nucleotide fragments were inserted into the pk18mob-pheS* plasmid us...

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Abstract

The invention discloses a rhodococcus gene editing method using a phenylalanyl-tRNA synthetase gene mutant as a reverse screening marker. The method forms a phenylalanyl-tRNA synthetase gene mutant by mutating the alanine site at the key site of the phenylalanyl-tRNA synthetase α-subunit in Rhodococcus to glycine, and the phenylalanyl-tRNA synthetase gene mutant is The tRNA synthesized by the tRNA synthetase mutant can assemble p-chlorophenylalanine into the protein, thereby inactivating the protein, leading to the death of the bacteria, and achieving the purpose of screening. Using the phenylalanyl-tRNA synthetase gene mutant as a reverse selection marker in Rhodococcus gene editing can realize efficient screening of successfully edited strains.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and relates to a Rhodococcus gene editing method using a phenylalanyl-tRNA synthetase gene mutant as a reverse selection marker. Background technique [0002] Rhodococcus belongs to Actinomycetes, Actinomycetes, Nocardiaceae, Rhodococcus, and is widely distributed in soil, rocks, groundwater, seabed sediments, insect offal and animal feces. Rhodococcus strains can utilize and degrade various forms of carbon sources, such as sugars, alkanes, and polyphenols, and especially have a strong degradation effect on some refractory heterocyclic compounds, such as pesticides, complex in petroleum Alkanes and lignin. In addition, due to many special enzymes and metabolic pathways in Rhodococcus, many valuable industrial products can be synthesized, such as acrylamide and other amide compounds, biosurfactants and bioflocculants (Hua Gougen, Guo Jianhua. Hong Research progress on the classification and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/90C12R1/01
CPCC12N15/902
Inventor 金明杰蔡成固许召贤
Owner NANJING UNIV OF SCI & TECH
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