Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes.
Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes.
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DOI：
10.1093/nar/gkl1158
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发表时间：
2007
影响因子：
14.9
通讯作者：
Huang JD
中科院分区：
文献类型：
--
作者：
Watt RM;Wang J;Leong M;Kung HF;Cheah KS;Liu D;Danchin A;Huang JD
为了研究在大肠杆菌中进行基因组规模的蛋白质标记和定位研究的可行性，选择了23个编码DNA序列（CDS）的代表性子集，用一个或多个荧光蛋白基因（EGFP、EYFP、mRFP 1、DsRed 2）进行染色体标记。我们使用λ-Red重组将PCR生成的含有荧光蛋白基因和抗生素抗性标记的DNA靶向盒精确有效地定位在感兴趣的CDS的C末端，在其天然启动子的控制下产生框内融合。我们结合了cre/loxP和flpe/frt技术，以使多轮染色体标记事件能够在对靶基因座的破坏最小的情况下顺序进行，从而允许蛋白质组在细胞内共定位。活E.大肠杆菌细胞使用荧光显微镜揭示了一个惊人的各种分布，包括：膜和类核协会，极灶和弥漫性细胞质定位。进行的52个独立靶向实验中有50个是成功的，并且23个选择的CDS中的21个可以荧光可视化。结果表明，E.大肠杆菌具有一个有组织的和动态的蛋白质组，并证明这种方法是适用于标签和（共）定位的CDS在全基因组范围内的规模。
To investigate the feasibility of conducting a genomic-scale protein labeling and localization study in Escherichia coli, a representative subset of 23 coding DNA sequences (CDSs) was selected for chromosomal tagging with one or more fluorescent protein genes (EGFP, EYFP, mRFP1, DsRed2). We used λ-Red recombination to precisely and efficiently position PCR-generated DNA targeting cassettes containing a fluorescent protein gene and an antibiotic resistance marker, at the C-termini of the CDSs of interest, creating in-frame fusions under the control of their native promoters. We incorporated cre/loxP and flpe/frt technology to enable multiple rounds of chromosomal tagging events to be performed sequentially with minimal disruption to the target locus, thus allowing sets of proteins to be co-localized within the cell. The visualization of labeled proteins in live E. coli cells using fluorescence microscopy revealed a striking variety of distributions including: membrane and nucleoid association, polar foci and diffuse cytoplasmic localization. Fifty of the fifty-two independent targeting experiments performed were successful, and 21 of the 23 selected CDSs could be fluorescently visualized. Our results show that E. coli has an organized and dynamic proteome, and demonstrate that this approach is applicable for tagging and (co-) localizing CDSs on a genome-wide scale.
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影响因子：
56.9
作者：
Daley, DO;Rapp, M;von Heijne, G
通讯作者：
von Heijne, G
影响因子：
64.8
作者：
Gavin, AC;Bösche, M;Superti-Furga, G
通讯作者：
Superti-Furga, G
影响因子：
5.6
作者：
Kholti, A;Charlier, D;Glansdorff, N
通讯作者：
Glansdorff, N
影响因子：
3.6
作者：
Davies, KM;Dedman, AJ;Lewis, PJ
通讯作者：
Lewis, PJ
DOI：
10.1073/pnas.0305747101
发表时间：
2004-01-20
影响因子：
11.1
作者：
Janakiraman, A;Goldberg, MB
通讯作者：
Goldberg, MB