Halobacterium NRC-1 oxygen, light and htlD perturbation. Halobacterium salinarum NRC-1

A 50ml culture of Halobacterium NRC-1 knockout strain delta-ura3 with additional in-frame gene deletion of htlD was grown to mid-log phase in a 125ml flask in rich media under varying oxygen and light conditions. Low and high light indicate growth in a painted flask versus growth in a standard clear flask under constant standard incubator illumination, respectively. Low and high oxygen indicate shaking in a stoppered flask at 100RPM agitation or shaking in a non-stoppered flask at 220RPM, respectively. Each of the four possible pairwise environemental combinations were assayed : High-oxygen/high-light, Low-oxygen/high-light, High-oxygen/low-light and Low-oxygen/low-light. RNA extractions were performed using the Stratagene Absolutely RNA Miniprep Kit and RNA quality checked with the Agilent Bioanalyzer and with Oligo Microarrays were fabricated at the Institute for Systems Biology Microarray Facility. The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1 Labeling, hybridization and washing have been previously described (Baliga et al. 2002) with 10 μg of RNA from the sample and reference. RNA from the final time point of a replicate experiment was used as the reference. Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip). Raw data was processed and converted into log10 ratios with lambda (λ) values determined by a maximum likelihood method.Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V. & Hood, L. (2002) Proc Natl Acad Sci U S A 99:14913-84. Keywords: environmental response, genetic perturbation Overall design: 4 samples were analyzed in duplicate (8 total microarrays) as dye-flips.

将携带Δura3敲除且额外带有htlD框内基因缺失的盐杆菌（Halobacterium）NRC-1敲除菌株的50mL培养液，置于125mL摇瓶中，于丰富培养基内、不同氧气与光照条件下培养至对数中期。其中，低光照与高光照条件分别指在涂覆避光层的摇瓶中培养，以及在标准透明摇瓶中于恒温培养箱恒定光照条件下培养；低氧与高氧条件分别指在封口摇瓶中以100转/分钟转速振荡培养，以及在敞口摇瓶中以220转/分钟转速振荡培养。四种两两组合的环境条件均进行了检测：高氧/高光照、低氧/高光照、高氧/低光照以及低氧/低光照。
RNA提取采用Stratagene Absolutely RNA微量提取试剂盒，RNA质量通过安捷伦生物分析仪（Agilent Bioanalyzer）检测；寡核苷酸微阵列由系统生物学研究所微阵列平台制备。该微阵列针对盐杆菌NRC-1的2400个非冗余基因，每个独特的70聚寡核苷酸设置4个重复点样。样本与参考RNA的标记、杂交及洗涤流程已在既往研究（Baliga等，2002）中发表，每反应使用10μg来自样本与参考的RNA。本研究以重复实验最后一个时间点的RNA作为参考样本。通过翻转标记荧光染料（dye-flip）校正染料掺入偏倚。原始数据经处理后转换为以10为底的对数比值（log₁₀比值），其中λ值通过最大似然法计算得到。
参考文献：Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V. & Hood, L. (2002) 美国国家科学院院刊, 99: 14913-14984。
关键词：环境应答、遗传扰动
实验设计：4份样本均进行重复检测（共8张微阵列芯片），采用染料互换（dye-flip）实验设计。