Recombinant cronobacter sakazakii and application thereof
A technology of bacilli and strains, which is applied in the fields of genetic engineering and biomedicine, can solve problems such as antibiotic insensitivity, and achieve the effects of good strain growth, single structure, and good growth
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Embodiment 1
[0058] Example 1: Construction of recombinant strain C. sakazakii ATCC BAA-894ΔRS18960
[0059] Specific steps are as follows:
[0060] 1. Obtaining the ESA_RS18960 gene knockout fragment
[0061] The ESA_RS18960 gene knockout plasmid was obtained by enzyme-cut ligation and overlapping PCR.
[0062] The two ends of the ESA_RS18960 gene knockout fragment are the upstream homology arm SEQ ID NO.4 of the ESA_RS18960 gene and the downstream homology arm SEQ ID NO.5, the middle is the Kan resistance gene SEQ ID NO.2 marker, the nucleus of the ESA_RS18960 gene knockout fragment The nucleotide sequence is shown in SEQ ID NO.3. The ESA_RS18960 gene knockout fragment was cloned into the plasmid pBlueScriptⅡSK(+) to obtain the knockout plasmid pBS-18960. Using the plasmid as a template, the knockout fragment of the ESA_RS18960 gene can be obtained by PCR.
[0063] Table 1: Knockout Primers
[0064] Primer Sequence (5'-3') Restriction sites 18960-U-F CGT CTCGAG...
Embodiment 2
[0074] Example 2: The specific steps of the MIC analysis of the recombinant strain C. sakazakii ATCC BAA-894ΔRS18960 on different antibiotics are as follows: (taking novobiocin as an example)
[0075] 1. Analysis of drug resistance of C. sakazakii ATCC BAA-894ΔRS18960 to novobiocin
[0076] (1) In a 96-well plate, different concentrations of novobiocin were added to each well by the double dilution method;
[0077] For each concentration, add 180 μL LB liquid medium plus 20 μL antibiotic stock solution to the first well, mix well and transfer 100 μL to the second well with 100 μL LB, then transfer 100 μL from the second well to add 100 μL LB in the third hole, and so on.
[0078] The strain was cultured in LB liquid medium overnight to mid-late logarithmic (OD 600 =3.00), the sample was diluted with fresh LB medium, and the bacterial concentration was OD 600 Dilute to 1 x 10 -3 , add 100 μL to the 96-well plate.
[0079] The last well was a blank control with no antibioti...
Embodiment 3
[0096] Example 3: Lipopolysaccharide Analysis of Recombinant Strain C. sakazakii ATCC BAA-894ΔRS18960
[0097] Lipopolysaccharide, also known as endotoxin, exists in most Gram-negative bacteria. It is a polysaccharide substance chimeric on the outside of the bacterial cell membrane. It is highly pathogenic. The structure of lipopolysaccharide (LPS) mainly consists of three Partial composition. From the inside to the outside, there are the hydrophobic lipid molecule A (Lipid A), the less variable core oligosaccharide (Coreoligosaccharide, OS), and the most variable part of the sugar chain structure O-antigen (O-antigen). Lipopolysaccharide plays an extremely important role in Gram-negative bacteria. It can increase the rigidity of cells by increasing the strength of cell walls, adapt to different external environments by inducing structural changes, and enable life to survive under different conditions. In addition, it can maintain the integrity of the bacterial membrane wall ...
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