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Re-engineering mediated sinorhizobium meliloti Rm1021 gene knockout method

A technology of Sinorhizobium, rm1021, applied in the field of genetic engineering, can solve the problems of cumbersome efficiency, time-consuming, highly inactive N-N health and so on

Inactive Publication Date: 2014-09-17
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

78% of the air is nitrogen, and nitrogen is an inert gas, the N-N bond is highly inactive, and it is difficult to convert nitrogen into effective ammonium salts by conventional chemical reactions
This classic gene knockout method is time-consuming (involving multiple gene cloning and sequencing), cumbersome (multiple strains and multiple manipulation steps), and inefficient (finally obtained clones may contain a large proportion of the original strain)

Method used

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  • Re-engineering mediated sinorhizobium meliloti Rm1021 gene knockout method
  • Re-engineering mediated sinorhizobium meliloti Rm1021 gene knockout method
  • Re-engineering mediated sinorhizobium meliloti Rm1021 gene knockout method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Construction of Recombinase Expression Plasmid

[0042] Design primer RN1:5'-GAA GAGCTCCATATG GATATTAATACTGAAACTG-3', (SEQ ID NO.1), the designed SacI and NdeI sites are underlined; RN2: 5'-GAA TCTAGA GTCATCGCCATTGCTCCCCAAATAC-3', (SEQ ID NO. 2), the designed XbaI site is underlined. Using lambda phage DNA (purchased from Shanghai Sangon Biological Co., Ltd.) as a template, RN1 and RN2 were PCR amplified to obtain a 1.9kb DNA fragment, digested with SacI and XbaI, and cloned into pBluescript II KS(-) digested with the same enzyme, After sequencing verification, the recombinant clone pNdRed was obtained. pNdRed was digested with NdeI and XbaI, and a 1.9kb DNA fragment was isolated, connected to the pSRKGm digested with the same enzyme, and 25 μg / ml gentamycin, IPTG and X-gal (5-bromo-4 chloro-3 -indole-β-D-galactoside) for screening. The white clone was picked and identified by plasmid digestion, and the recombinant clone pLS2405 was obtained. pKsacB wa...

Embodiment 2

[0043] Example 2. Knockout of the bacA gene on the S. meliloti Rm1021 genome

[0044] 1. Preparation of Rm1021 electroporation competent cells and transformation of pLS2406

[0045] Streak Rm1021 into LBMC solid medium from the cryopreservation tube at -80°C, and culture at 30°C for about 60 hours. Transfer a single colony to 3ml LBMC, shake culture at 30°C, 220rpm for about 36h, then transfer 2ml to 100ml LBMC, adjust the OD600 to about ~0.1, continue to culture until the OD600 is about 0.6, centrifuge, and discard the supernatant. The cell pellet was washed three times with ice-cold 10% glycerol, finally suspended in 200 μl of ice-cold 10% glycerol, and distributed in 50 μl. Add 100ng of pLS2406 to the competent cells, flick and mix well, transfer to a 1mm electric cup cooled on ice, and transform with 2.1kV electric shock, the electric transformation instrument is the Gene Pulser of Bio-Rad, USA II . After electrotransformation, add 1ml of SOC to suspend, and incubate at...

Embodiment 3

[0059] Example 3. Knockout of the exoR gene on the S. meliloti Rm1021 genome

[0060] The strategy of exoR gene knockout is the same as that of bacA. Design three pairs of primers R1341: 5'-TATCCTGACCAGCGGCAGTTC-3', (SEQ ID NO.13), R1342: 5'-GTGCGCGGAACCCCTATTTGTTCATAACAATCAGTTTCTTTCGTTC-3', (SEQ ID NO.14); R1343: 5'-GAACGAAAGAAACTGATTGTTATGAACAAATAGGGGTTCCGCGCAC-3' (SEQ ID NO. 15), R1344: 5'-GTCAGGCCGCACGGGACCTCATCCTTAGTTCCTATTCCGAAGTTC-3', (SEQ ID NO. 16); R134: 5'-GAACTTCGGAATAGGAACTAAGGATGAGGTCCCGTGCGGCCTGAC-3', (SEQ ID NO. 17), R1346: 5'-GGAAAAGACCCCCTA -3', (SEQ ID NO. 18). Wherein R1342 and R1343 and R1344 and R1345 are reverse complementary sequences, which are the overlapping sequence parts between the templates of the second step OE-PCR.

[0061] Using Rm1021 genomic DNA as a template, R1341 and R1342 PCR, R1325 and R1326 were used to amplify the exoR gene upstream and downstream respectively 0.5 kb; using pLS1918 as a template, R1343 and R1344 PCR amplified a 1.0 ...

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Abstract

The invention relates to a re-engineering mediated sinorhizobium meliloti Rm1021 gene knockout method. The method comprises the following steps: firstly, carrying out amplification via chain reactions of overlapping-extending polymerases so as to obtain homologous fragments which have about 500bp and aim at genes to be knocked out at two sides and a combined DNA fragment of kanamycin resistance genes at the middle; secondly, electrically converting the DNA fragment into a cell Rm1021 expressed by recombinase induced by isopropyl-Beta-D-sulfo-galactoside; replacing target genes with the kanamycin resistance genes under the kanamycin resistance screening so as to obtain a gene knockout mutation strain; and finally, culturing the strain in a solid culture medium containing 0.4% of saccharose so as to eliminate plasmids containing recombinase genes. The adopted ecombinase genes are derived from Lambda phages and cloned on plasmids pLS2406. Meanwhile, the plasmids pLS2406 contain negative-screening marked sacB genes.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for knocking out the Rm1021 gene of Sinorhizobium meliloti mediated by recombinant engineering. Background technique [0002] With the development of modern society, the energy problem has become an increasingly prominent worldwide problem, and the demand for energy in various countries is becoming more and more urgent. How to obtain the energy existing in nature most efficiently is one of the keys in the field of energy research and development. Nitrogen source is an important resource, and ammonium salt whose main component is nitrogen occupies a large proportion in agricultural feed and fertilizer. 78% of the air is nitrogen, and nitrogen is an inert gas. The N-N bond is highly inactive, and it is difficult for conventional chemical reactions to convert nitrogen into effective ammonium salts. Therefore, how to effectively use the nitrogen resources in the air is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/20C12R1/41
Inventor 尚广东陈中秋庄浩李玲
Owner NANJING NORMAL UNIVERSITY
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