A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery.
A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery.
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DOI：
10.7554/elife.69549
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发表时间：
2021-08-03
期刊：
影响因子：
7.7
通讯作者：
Rudolf F
中科院分区：
文献类型：
--
作者：
Azizoglu A;Brent R;Rudolf F
基因的条件表达和表型的观察仍然是生物学发现的核心。目前的方法能够实现开/关或不精确控制的分级基因表达。我们开发了一个“脾气好”的控制器，WTC 846，精确可调，分级，生长条件的独立表达的基因在酿酒酵母。受控基因从一个强的半合成启动子表达，该启动子被原核TetR抑制，TetR也抑制其自身的合成;基础表达被第二个“归零”抑制子废除。自动阻遏环降低细胞间的变异，同时能够通过化学诱导剂精确调节蛋白质表达。WTC 846等位基因菌株，其中控制器取代了天然启动子重演已知的无效表型（CDC 42，TPI 1），表现出新的过表达表型（IPL 1），显示蛋白质剂量依赖性的生长速率和形态表型（CDC 28，TOR 2，PMA 1和迄今未表征的PBR 1），并使细胞周期同步（CDC 20）。WTC 846定义了一个“表达钳”，允许实验者在细胞蛋白丰度范围内调整蛋白剂量，并在设定点附近进行有限的变化。
Conditional expression of genes and observation of phenotype remain central to biological discovery. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a 'well-tempered' controller, WTC846, for precisely adjustable, graded, growth condition independent expression of genes in Saccharomyces cerevisiae. Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which also represses its own synthesis; with basal expression abolished by a second, 'zeroing' repressor. The autorepression loop lowers cell-to-cell variation while enabling precise adjustment of protein expression by a chemical inducer. WTC846 allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes (CDC42, TPI1), exhibited novel overexpression phenotypes (IPL1), showed protein dosage-dependent growth rates and morphological phenotypes (CDC28, TOR2, PMA1 and the hitherto uncharacterized PBR1), and enabled cell cycle synchronization (CDC20). WTC846 defines an 'expression clamp' allowing protein dosage to be adjusted by the experimenter across the range of cellular protein abundances, with limited variation around the setpoint.
影响因子：
9.9
作者：
Pesce CG;Zdraljevic S;Peria WJ;Bush A;Repetto MV;Rockwell D;Yu RC;Colman-Lerner A;Brent R
通讯作者：
Brent R