Guo H N, Chen Z, Xiang J
Department of Burns and Plastic Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China (is working at the Department of Burns, Zhengzhou First People's Hospital, Zhengzhou 450004, China).
Department of Burns and Plastic Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China(is working at the Department of Anesthesiology, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China).
Zhonghua Shao Shang Za Zhi. 2020 Jan 20;36(1):32-36. doi: 10.3760/cma.j.issn.1009-2587.2020.01.006.
To investigate the influence of gene knockout on growth metabolism and biofilm formation of . The gene was knocked out from standard strain ATCC 17978 (wild strain) by homologous recombination method, and then the ATCC 17978 knockout strain (ATCC 17978/ΔabaR: : Kn) was obtained and verified by polymerase chain reaction (PCR) electrophoresis and sequencing. The growth curves of wild strain and knockout strain were determined by microplate reader within cultivation hour (CH) 18, and the biofilm formation ability was measured by crystal violet staining at CH 8, 24, and 48, respectively. The sample number at each time point was 3.The results were denoted as absorbance value. Data were processed with analysis of variance of factorial design, one-way analysis of variance, test, and least-significant difference test. (1) The length of PCR product of target fragment ΔabaR: : Kn was 3 029 bp. The gene was knocked out to obtain the knockout strain ATCC 17978/ΔabaR: : Kn. The length of PCR product of the knockout strain was 3 300 bp. The gene was successfully knocked out. (2) At CH 2, 3, and 4, the absorbance values of wild strain were slightly higher than those of the knockout strain. The absorbance values of wild strain and knockout strain were similar from CH 5 to 18. (3) At CH 8 and 24, the biofilm formation ability of wild strains (0.644±0.066, 0.574±0.184) was similar to that of knockout strains (0.559±0.008, 0.394±0.030, =2.209, 1.167, >0.05). At CH 48, the biofilm formation ability of wild strains (1.157±0.259) was significantly stronger than that of knockout strains (0.576±0.026, =3.865, <0.05). The biofilm formation ability of wild strains at CH 48 was significantly stronger than that at CH 8 and 24 (<0.05). The biofilm formation ability of knockout strains at CH 24 was significantly weaker than that at CH 8 and 48 (<0.05). The gene of ATCC 17978 can be successfully knocked out by homologous recombination to obtain its knockout strain ATCC 17978/ΔabaR: : Kn. The gene does not affect the growth and metabolism of but can weaken its biofilm formation ability.
为研究基因敲除对……生长代谢和生物膜形成的影响。采用同源重组方法从标准菌株ATCC 17978（野生菌株）中敲除该基因，随后获得ATCC 17978基因敲除菌株（ATCC 17978/ΔabaR::Kn），并通过聚合酶链反应（PCR）电泳和测序进行验证。使用酶标仪在培养18小时内测定野生菌株和基因敲除菌株的生长曲线，分别在培养8小时、24小时和48小时通过结晶紫染色测定生物膜形成能力。每个时间点的样本数为3。结果以吸光度值表示。数据采用析因设计方差分析、单因素方差分析、t检验和最小显著差检验进行处理。（1）目标片段ΔabaR::Kn的PCR产物长度为3029 bp。该基因被敲除后获得基因敲除菌株ATCC 17978/ΔabaR::Kn。基因敲除菌株的PCR产物长度为3300 bp。该基因成功被敲除。（2）在培养2小时、3小时和4小时时，野生菌株的吸光度值略高于基因敲除菌株。从培养5小时到18小时，野生菌株和基因敲除菌株的吸光度值相似。（3）在培养8小时和24小时时，野生菌株（0.644±0.066，0.574±0.184）的生物膜形成能力与基因敲除菌株（0.559±0.008，0.394±0.030，t=2.209，1.167，P>0.05）相似。在培养48小时时，野生菌株（1.157±0.259）的生物膜形成能力显著强于基因敲除菌株（0.576±0.026，t=3.865，P<0.05）。野生菌株在培养48小时时的生物膜形成能力显著强于培养8小时和24小时时（P<0.05）。基因敲除菌株在培养24小时时的生物膜形成能力显著弱于培养8小时和48小时时（P<0.05）。ATCC 17978的该基因可通过同源重组成功敲除，获得其基因敲除菌株ATCC 17978/ΔabaR::Kn。该基因不影响……的生长和代谢，但会削弱其生物膜形成能力。
