Halobacterium NRC-1 oxygen, light and phr2 perturbation. Halobacterium salinarum NRC-1

A 50ml culture of Halobacterium NRC-1 knockout strain delta-ura3 with additional in-frame gene deletion of phr2 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.

将一株携带额外框内phr2基因缺失的盐杆菌（Halobacterium）NRC-1 delta-ura3敲除菌株的50mL培养物，置于125mL烧瓶的富培养基中，在不同氧气与光照条件下培养至对数中期。其中，低光照与高光照分别指在涂漆烧瓶中培养，以及在恒定标准培养箱光照下的标准透明烧瓶中培养；低氧与高氧分别指在塞紧的烧瓶中以100转/分钟摇振培养，或在未塞紧的烧瓶中以220转/分钟摇振培养。共计四种两两组合的环境条件均完成检测：高氧/高光照、低氧/高光照、高氧/低光照与低氧/低光照。RNA提取采用Stratagene公司Absolutely RNA微量提取试剂盒，RNA质量通过Agilent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（Institute for Systems Biology）微阵列设施制备。该微阵列针对盐杆菌（Halobacterium）NRC-1的2400个非冗余基因，每个独特的70聚体寡核苷酸设有4个重复点。标记、杂交与洗涤步骤已在既往研究中详述（Baliga等，2002），实验中每份样品与参考样本均使用10μg RNA。以重复实验最后一个时间点的RNA作为参考样本。通过反转标记荧光染料（染料互换，dye-flip）校正染料掺入偏差。原始数据经处理后转换为以10为底的对数比值，其中λ值通过最大似然法计算得到。Baliga, N. S.、Pan, M.、Goo, Y. A.、Yi, E. C.、Goodlett, D. R.、Dimitrov, K.、Shannon, P.、Aebersold, R.、Ng, W. V.与Hood, L.（2002），《美国国家科学院院刊》，99卷：14913-14984。关键词：环境响应，遗传扰动。实验设计：4份样本均进行重复检测（共计8张微阵列），采用染料互换（dye-flip）策略。