Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi
Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi
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DOI：
10.1128/aem.02454-06
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发表时间：
2007-03-01
影响因子：
4.4
通讯作者：
Norgard, Michael V.
中科院分区：
文献类型：
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作者：
Blevins, Jon S.;Revel, Andrew T.;Norgard, Michael V.
开发新的遗传系统来研究伯氏疏螺旋体内发生的复杂调控事件是当代莱姆病研究的重要目标。虽然最近在伯氏杆菌的遗传操作方面取得了一些进展，但仍然缺乏基本的分子系统来评估这种病原菌的差异基因表达。在这里，我们描述了两个强大的遗传工具的适应用于伯氏杆菌。第一个是红叶石楠萤火虫荧光素酶基因报告，它是密码子优化的，以增强在B.burgdorferi的翻译。利用这一修饰的报告基因，我们证明了在新鲜兔血的存在下，将编码外表面蛋白C(OSPC)启动子的穿梭载体与荧光素酶报告基因融合在一起，转化伯氏杆菌后，荧光素酶的表达增加。第二种是优化的紫胶操作者/抑制器系统，以达到最严格的监管程度。使用上述荧光素酶报告，我们评估了异丙基-对-D-硫代半乳糖苷(IPTG)依赖的基因表达的动力学和最大水平。该乳糖诱导表达系统还用于表达1p25携带的丝状螺旋体持续存在所需基因(BPTA)。这些进展应该普遍适用于进一步评估伯氏杆菌毒力表达中可能涉及的其他基因的调控。
The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-p-D-thiogalactopyranoside (IPTG) -dependent gene expression. This lac-inducible expression system also was used to express the gene carried on 1p25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.