1.2 MRSA cultivation
The standard strain of Staphylococcus aureus ATCC25923 used by the
Institute was purchased from the bacterial library of the Chinese
Academy of Sciences.50 MRSA and 50 KO sdrc (purchased from Beijing Abace
Biotechnology Co. LTD ) stored at -80 ℃ each μ Add 15ml of soybean broth
culture medium, incubate overnight at 37 ℃ at 200r/min, and detect the
knockout efficiency of sdrc using RT-PCR. Take 5 out of MRSA and KO sdrc
recovered overnight μ Add 5ml of soybean broth medium and culture on a
constant temperature shaker. Measure the absorbance value of OD600 at
1h, 2h, 3h, 4h, 5h, 7h, 9h, 12h, 18h, and 24h according to: <i
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plot bacterial growth curves.
1.3 ELISA experiment
Collect samples and perform enzyme-linked immunosorbent assay according
to the manufacturer’s protocol, including α- HL, PSM, CRP, TNF- α、
IL-6.
1.4 Crystal violet staining
Remove the bacteria cultured overnight from the culture medium and add
500-1000 to it μ Place 4% paraformaldehyde at room temperature for
30-40 minutes, remove excess liquid, tilt the plate, dry at 37 ℃ for 1
hour, add crystal violet that can cover the bottom of the hole, stain at
room temperature for 20 minutes, add PBS to wash 2-3 times, and absorb
the remaining liquid to observe the ability of biofilm formation.
1.5 Observation of MRSA biofilm formation using scanning electron
microscopy
MRSA and KO sdrc were cultured overnight at 37 ℃ in a 24 well plate and
placed under a scanning electron microscope to observe bacterial
morphology.
1.6 RT-PCR experiment
Extract total RNA from each group separately, then perform reverse
transcription reaction, and then use 2 x Universal Blue SYBR Green qPCR
Master Mix reagent kit for qPCR reaction. The reaction procedure is: pre
denaturation at 95 ℃ for 1 minute; 95 ℃ denaturation for 20 seconds, 55
℃ annealing for 20 seconds, 72 ℃ extension for 30 seconds; 40 cycles.
The primer sequence is shown in Table 1:
Table 1 The sequences of the Primers