Escherichia coli MG1655 bacterial strain lacking sahn gene, construction method and application

Abstract

The invention relates to an Escherichia coli MG1655 bacterial strain lacking a sahn gene, a construction method and application. The construction method comprises the following steps that a linear DNA fragment of which the middle part is a selection marker gene and the two ends are homologous sequences is utilized, by means of the effect of a Red recombinase, a homologous recombination between the homologous sequences and a desired gene occurs, and thus a target gene is replaced with the marker gene; in order to eliminate the trace of the selection marker gene, special sites which can be recognized by a Cre site specificity recombinase are added on the both sides of the selection marker gene; and the selection marker gene is deleted through the effect of the site specificity recombinase, and the Escherichia coli MG1655 bacterial strain lacking the sahn gene, namely MG1655 delta sahn, is obtained. The invention further provides the application of the bacterial strain. The quorum sensing characteristic of the mutant strain delta sahn is utilized as a contrast, and a novel type inhibitor which is 1-((4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methyl)-5-(1H-1,2,3-triazo1-4-yl)piperidin-3-ol is screened, so that the growth and splitting ability of pathogenic bacterium is reduced.

Description

[0001] Technical field: [0002] The invention relates to the field of bioengineering, in particular to an Escherichia coli MG1655 strain lacking sahn gene, its construction method and application. [0003] Background technique: [0004] Altering the structure of antimicrobial drug targets through methylation modification is an important drug resistance mechanism of pathogenic bacteria represented by Escherichia coli. S-adenosylhomocysteine ​​nuclease (SAHN) expressed by sahn gene in Escherichia coli genome is involved in the methyl cycle as an important enzyme in the methylation reaction regulated by S-adenosylmethionine (SAM) Regenerates methionine and adenine. The inhibition of SAHN enzyme activity in the cell will seriously affect the growth and division of bacteria. The reason is that, first of all, the decrease of SAHN enzyme activity will cause the accumulation of S-adenosylhomocysteine ​​(SAH) in the bacteria, which acts as a formazan on which SAM depends. As an effec...

AI Technical Summary

Problems solved by technology

The inhibition of SAHN enzyme activity in the cell will seriously affect the growth and division of bacteria. The reason is that, first of all, the decrease of SAHN enzyme activity will cause the accumulation of S-adenosylhomocysteine ​​(SAH) in the bacteria, which acts as a formazan on which SAM depends. As an effective feedback inhibitor of base transferase, the accumulation of SAH in the bacteria will completely inhibit the methyl transfer reaction, and then reduce the adaptability of pathogenic bacteria by affecting the regulation of gene expression and protein modification, so that the growth and division of bacteria can be improved. Inhibition; secondly, the inhibition of SAHN enzyme will cause the loss of AI-2 precursor of the downstream quorum sensing effect (QS) signal molecule, and the pathogenic bacteria cannot synthesize the AI-2 signal molecule, which can effectively control the expression of pathogenic bacteria virulence factors, biofilm formation, etc. Bacterial quorum sensing effect; moreover, the inhibition of SAHN enzyme will cause the interruption of the methyl cycle, and the important compounds in the methyl cycle, methionine and SAM, cannot be regenerated, and SAM cannot pass through the bacterial cell membrane. Without the supply of methionine, pathogenic bacteria cannot grow at all

The deletion of the bacterial sahn gene will result in the inability to express S-adenosyl homocysteine ​​nuclease (SAHN), which will affect the growth and division of bacteria

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