乳糖阻遏蛋白温度敏感型突变体（lacIts）和乳糖阻遏蛋白超阻遏温度敏感型突变体（lacIqts）表达质粒的构建及温度敏感型乳糖阻遏蛋白的评估

Construction of lacIts and lacIqts expression plasmids and evaluation of the thermosensitive lac repressor.

作者信息

Hasan N, Szybalski W

机构信息

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706-1599, USA.

出版信息

Gene. 1995 Sep 22;163(1):35-40. doi: 10.1016/0378-1119(95)00409-y.

DOI:10.1016/0378-1119(95)00409-y

PMID:7557475

Abstract

To characterize a thermosensitive lacIts mutant isolated by Bukrinsky et al. [Gene 70 (1989) 415-417] and to adapt it as a convenient control element, we have (i) mapped the mutation to the inducer-binding domain of the LacIts repressor, (ii) determined that the mutation resulted in the loss of a HaeIII site in lacIts due to a G-->A transition, leading to a Gly187-->Ser substitution, (iii) removed extraneous lacZop DNA downstream of lacI, and (iv) cloned lacIts (in plasmids based on ori of either ColE1 or P15A) under control of the wild-type or lacIq promoters. The LacIts repressor is insensitive to IPTG. The repression of lacZop by LacIts is very efficient at 30 degrees C and total induction was achieved at 42 degrees C, providing that the LacIts concentrations are not excessive and that repressor-to-operator ratios are not too high.

摘要

为了表征Bukrinsky等人[《基因》70 (1989) 415 - 417]分离出的热敏性LacIts突变体，并将其作为一个方便的控制元件加以应用，我们进行了以下操作：(i) 将该突变定位到LacIts阻遏物的诱导剂结合结构域；(ii) 确定该突变由于G→A转换导致LacIts中一个HaeIII位点缺失，从而导致Gly187→Ser取代；(iii) 去除lacI下游多余的lacZop DNA；(iv) 在野生型或lacIq启动子的控制下克隆LacIts（在基于ColE1或P15A ori的质粒中）。LacIts阻遏物对IPTG不敏感。在30℃时，LacIts对lacZop的阻遏非常有效，并且在42℃时实现了完全诱导，条件是LacIts的浓度不过高且阻遏物与操纵基因的比例不太高。