Regulation of Gene Expression via Toehold Switch in E. coli
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摘要: 设计了一种核糖体调节器—“立足点开关”（toehold switch）。与传统核糖体调节器设计不同的是，该核糖体调节器的起始密码子（AUG）和核糖体结合位点位于核糖体调节器中发夹结构RNA的环（loop）上，而“茎”（stem）结构是完全互补配对的RNA双链。通过RNA链替换反应，引发链（trigger）RNA能够打开发夹结构RNA，从而激活下游绿色荧光蛋白的表达，导致荧光信号的增长，最终实现对大肠杆菌基因表达的调控。系统研究了“茎”的长度对绿色荧光蛋白表达的调控作用。实验结果表明，当“茎”的长度大于8个碱基时，发夹结构RNA就能有效地抑制绿色荧光蛋白的表达。进一步的共表达实验结果表明，引发链RNA能够打开发夹RNA，从而调控大肠杆菌基因表达。Toehold开关调控的大肠杆菌基因表达系统具有可拓展性，可应用于多基因表达调控，对基因疾病诊疗具有潜在应用价值。Abstract: A riboregulator called toehold switch has been designed. In this riboregulator, the start codon AUG and ribosome binding site (RBS) are located on the loop of the hairpin structure, resulting in the stem structure with a completely complementary double-stranded RNA. Trigger RNA can open the hairpin through strand displacement reaction, and activate the downstream expression of green fluorescence protein (GFP), leading to a certain increasing of fluorescence signal, eventually realizing the regulation of E. coli gene expression. The effects of stem length on the expression of GFP were investigated. Results showed that the hairpin structure of toehold switch could effectively inhibit the expression of green fluorescent protein when the length of stem was more than 8 bp. Further co-expression experiments showed that trigger RNA could open the structure of hairpin RNA, resulting in a certain increasing of expression of GFP, and regulate the gene expression of E. coli. The E. coli gene expression system regulated by toehold switch is scalable and can be applied to the regulation of multi-gene expression, which has potential application value in the diagnosis and treatment of gene diseases.
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Key words:
- riboregulator /
- toehold switch /
- hairpin RNA /
- stem /
- RNA strand displacement reaction
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图 1 （a）经典toehold switch结构及原理示意图；（b）流式细胞仪测量的荧光强度对比图
Figure 1. (a) Schematic diagram of classical toehold switch riboregulator; (b) Comparison of fluorescence intensity for different experimental samples measured by flow cytometry
图 2 switch RNA和trigger RNA的结构示意图：（a）核糖体结合位点RBS和AUG均在switch RNA中发夹结构的环上；（b）不同长度stem结构的toehold switch结构示意图
Figure 2. Schematic diagram of the structure of switch RNA and trigger RNA: (a) Both the ribosome binding site RBS and the start codon AUG are integrated on the loop of switch RNA; (b) Structures of toehold switch with stem of different lengths
图 3 流式细胞仪测量的switch RNA抑制基因表达荧光强度
Figure 3. Fluorescence intensity for switch RNA inhibitory gene expression measured by flow cytometry
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