| Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes
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Author:
Date:
2021-03-05
[Abstract] Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant library in traditional TIS, the defined mutant library sequencing (DML-Seq) has advantages as: 1) efficient mutagenesis; 2) low bottleneck effects; 3) avoid hotpots caused by screening; 4) can be directly used in the following experiments. Here, we described an optimized procedure of DML-Seq for fitness screen to supply classical TIS using the marine pathogenic bacterium Edwardsiella piscicida as an example.
[摘要] [摘要]转座子插入测序（TIS）是一项新兴技术，它利用大量的转座子突变体文库筛选特定表型，并结合高通量并行测序技术，在不同条件下确定细菌适应性的条件性基本遗传要求。与传统TIS中的大规模突变文库相比，已定义的突变文库测序（DML-Seq）具有以下优势：1）高效诱变；2）瓶颈效应低；3）避免因筛选引起的火锅；4）可直接用于以下实验。在这里，我们描述DML-SEQ的优化过程进行健身屏幕使用海洋致病菌提供古典TIS爱德华piscicida作为一个例子。
[背景]转座子插入诱变与下一代测序（NGS）结合已被证明是在多种条件下研究基因功能的有效方法（Chao等，2016； Price等，2018）。通常，TIS分析由转座子插入位点的大规模平行测序和大量插入事件的统计分析组成。
基于TIS的筛选可以在多种条件下基于高度饱和的转座子突变体文库的细菌适应性，提供单个基因座和域的适应性贡献的高分辨率图（Chao等，2016）。每个位点的插入频率或相应突变体的相对丰度通常与施加选择性压力（例如宿主和抗生素施加的压力）后与基因座对适应性的贡献成反比（Chao等人，2016）。这种方法的原理是多种相关方法的基础，包括TIS，转座子测序（TnSeq ），插入测序（INSeq ），转座子定向插入位点测序（TraDIS ...
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| The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein
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Author:
Date:
2020-08-05
[Abstract] ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged ...
[摘要] [摘要]ATP酶是将ATP分解为ADP并释放无机磷（Pi）的酶。在这里，我们提供了一个详细的方案来测定重组AAA+-ATPase蛋白（一般对照非抑制-4[GCN4]）的ATPase活性，在360nm处测定无机磷的累积量。一般而言，底物2-氨基-6-巯基-7-甲基嘌呤核苷（甲基硫代鸟苷，鸟苷类似物：MESG）在Pi存在下通过嘌呤核苷磷酸化酶（PNP）酶转化为核糖1-磷酸和2-氨基-6-巯基-7-甲基嘌呤。测量了由于酶转化而导致的MESG底物和随后的转化产物在330nm处的最大吸收度的分光光度偏移。将gcn4his标记的重组蛋白在大肠杆菌BL21细胞中表达，并用Ni-NTA柱纯化。将纯化后的蛋白质用于溶液中Pi的定量测定或连续测定由于GCN4的atp酶活性而释放的Pi，GCN4是一种在许多真核生物中保存的AAA+-ATPase蛋白，在植物受到生物和非生物胁迫时调节气孔开度。
[背景] ...
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