Study of Cj1501 function in Campylobacter jejuni

In order to study the function of the Campylobacter jejuni Cj1501 gene, a series of experiments were carried out. Three strains were constructed: a Cj1501 knockout strain, a strain where the Cj1501 knockout was complemented in trans, and a strain with a second copy over-expressing Cj1501 from an fdxA promoter. The transcriptomes of these were all compared to the wild-type strain. The arrays are all from RNA isolated in mid-exponential growth from independent biological replicates. Batch cultures of Campylobacter jejuni NCTC 11168 were grown in 50 ml volumes of Brucella broth in 70cm tissue culture flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (5% Oxygen, 10% Carbon dioxide, 85% Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 37 ºC. RNA was extracted from C. jejuni cultures grown to an OD600 of approximately 0.4. Briefly, 0.1 volume of 5% phenol in ethanol was mixed with the broth culture, and after centrifugation RNA was isolated with Tri Reagent (Sigma) and chloroform. RNA was further purified using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. The RNA was treated with Turbo DNA-free (Ambion) to remove any residual DNA according to the manufacturer’s instructions. RNA concentration was determined using the Nanodrop Spectrophotometer NS-1000 (Thermo Scientific). Two or three independent RNA preparations (biological replicates) of each sample type were labelled and hybridized to custom-designed Agilent microarrays. Equal quantities of RNA from test and control cultures were labelled by using nucleotide homologues of dUTP containing either Cy3 or Cy5 fluorescent dye (Perkin Elmer). For each microarray slide, the test strain was labelled with Cy3-dUTP, while the wild-type sample was labelled with Cy5-dUTP. RNA (15 µg) was primed with 5 µg pd(N)6 random hexamers (Amersham Biosciences). The Affinity Script kit (GE Healthcare) was used to produce cDNA, and hybridized (Agilent Hi-RPM Gene Expression Hybridization Kit) to the microarray slide according to the manufacturer’s instructions. Microarrays were scanned at 5 µm using an Axon 4000A scanner, and images were acquired using GenePix Pro 3.0 software (Axon). Related analysis reference: Holmes K., Mulholland F., Pearson B. M., Pin C., McNicholl-Kennedy J., Ketley J. M., Wells J. M. (2005) Campylobacter jejuni gene expression in response to iron limitation and the role of Fur Microbiology-SGM 151 243-257 (PMID 15632442).

为探究空肠弯曲菌（Campylobacter jejuni）Cj1501基因的功能，本研究开展了一系列实验。共构建三株工程菌株：分别为Cj1501基因敲除菌株、反式互补Cj1501敲除菌株，以及携带第二拷贝并通过fdxA启动子过表达Cj1501的菌株。将上述三株菌株的转录组与野生型菌株进行对比分析。
本实验的基因芯片数据均来自于对数中期生长阶段、独立生物学重复样本所提取的RNA。空肠弯曲菌NCTC 11168的批量培养采用70cm²细胞培养瓶，每瓶盛放50mL布鲁氏菌肉汤（Brucella broth）。培养环境采用英国Don Whitley Scientific公司生产的MACS-VA500微需氧工作站营造，该工作站可维持培养环境为5%氧气、10%二氧化碳、85%氮气，并将生长温度稳定控制在37℃。
当培养物的OD₆₀₀值达到约0.4时，收集空肠弯曲菌菌体并提取RNA。简要步骤如下：向肉汤培养液中加入0.1倍体积的5%苯酚乙醇溶液，离心后使用Tri Reagent（Sigma）与氯仿分离RNA。随后按照制造商说明书，使用RNeasy试剂盒（Qiagen）对RNA进行进一步纯化。按照制造商指导方法，使用Turbo DNA-free（Ambion）处理RNA以去除残留的基因组DNA。使用Nanodrop NS-1000分光光度计（Thermo Scientific）测定RNA浓度。
每种样本类型设置2~3个独立的RNA制备样本（即生物学重复），将其标记后与定制化安捷伦（Agilent）基因芯片进行杂交。取等量的待测样本与对照样本的RNA，使用带有Cy3或Cy5荧光染料的dUTP核苷酸类似物（Perkin Elmer）进行标记。每张基因芯片玻片上，待测菌株的RNA样本用Cy3-dUTP标记，野生型样本则用Cy5-dUTP标记。取15μg RNA，加入5μg pd(N)₆随机六聚体引物（Amersham Biosciences）进行引物退火。使用Affinity Script试剂盒（GE Healthcare）反转录合成cDNA，随后按照制造商说明书，使用安捷伦Hi-RPM基因表达杂交试剂盒（Agilent Hi-RPM Gene Expression Hybridization Kit）将cDNA与基因芯片玻片进行杂交。
使用Axon 4000A扫描仪以5μm分辨率扫描基因芯片，并用GenePix Pro 3.0软件（Axon）采集图像。
相关分析参考文献：Holmes K.、Mulholland F.、Pearson B. M.、Pin C.、McNicholl-Kennedy J.、Ketley J. M.、Wells J. M.（2005）空肠弯曲菌对铁限制的基因表达响应及Fur蛋白的调控作用，《Microbiology-SGM》，151卷，243-257页（PMID：15632442）。