Orthogonal riboswitches as tools for controlling gene expression in bacteria

基因表达是DNA序列信息首先转录成信使RNA（mRNA），然后翻译成蛋白质的过程。基因的表达受到许多机制的严格控制（调节），这些机制影响基因产物（RNA和蛋白质）响应环境条件和其他信号合成的时间和水平。例如，最近发现细胞中的某些代谢物可以通过触发mRNA中存在的开关来打开或关闭基因表达。这些所谓的核糖开关存在于生命的所有领域，尤其是在细菌中广泛传播。通常，这些开关调节特定代谢物的产生、降解或转运。例如，腺苷酸核糖开关结合腺嘌呤以激活编码腺嘌呤脱氨酶的mRNA的翻译，该酶降解腺嘌呤。通过这种反馈机制，细菌可以控制细胞水平的腺嘌呤，这是DNA合成的关键组成部分。最近，我们成功地重新设计（或重新布线）添加A-核糖开关，使它们不再由细胞中存在的天然代谢物触发，而是可以通过添加各种合成分子（配体）来控制。在这个项目中，我们的目标是开发新的正交核糖开关，通过进一步的遗传操作和开发新的和更有效的合成配体，这将允许更精确和动态控制的基因表达在细菌中。除了可以激活结合合成配体的基因表达的核糖开关外，我们还将应用我们的策略来重新设计核糖开关，其可以响应选定的合成分子来阻断基因表达。我们还将把多个开关串联在一起，以实现对基因表达的更数字化控制。例如，我们建议开发一种基因开关，它可以用一种配体打开，激活基因表达，然后用第二种不同的配体关闭。此外，我们还将展示如何相互正交的核糖开关可以用来影响细菌中多个基因的同时和差异控制。我们开发的新基因开关可用于研究细菌中基因的功能。在致病细菌的情况下，基于正交核糖开关的表达工具可用于验证抗生素的新靶点，从而开发新的抗菌治疗方法。核糖开关表达工具也可用于帮助细菌中蛋白质的产生，其可包括治疗上重要的蛋白质（生物药物）或工业上重要的酶（生物催化剂）。此外，正交核糖开关可用于控制编码代谢途径的多个基因，这些代谢途径导致基于天然产物的药物、生物燃料和其他商业上重要的产品，这些产品正在成为合成生物学的靶标。在这个项目中，我们将致力于开发具有所需特性的正交核糖开关，这将使我们能够证明它们在这些重要应用中的实用性。

Gene expression is the process by which DNA sequence information is first transcribed in to messenger RNA (mRNA), and then translated to produce proteins. The expression of genes is tightly controlled (regulated) by a number of mechanisms, which affect the timing and levels by which the gene products (RNAs and proteins) are synthesised in response to environmental conditions and other signals. For example, it was recently discovered that certain metabolites in cells can turn gene expression on or off by triggering switches present within mRNA. These so called riboswitches are found in all domains of life, and are particularly wide spread in bacteria. Typically these switches regulate the production, degradation or transport of specific metabolites. For example the add A-riboswitch binds adenine to activate translation of the mRNA that encodes the enzyme adenine deaminase, which degrades adenine. Through this feedback mechanism the bacteria can control the cellular levels of adenine a key building block in DNA synthesis. Recently we succeeded in re-engineering (or rewiring) add A-riboswitches, so that they are no longer triggered by the natural metabolites present in the cell, but instead can be controlled by the addition of various synthetic molecules (ligands). In this project we aim to develop new orthogonal riboswitches through further genetic manipulation and by developing new and more effective synthetic ligands which will allow more precise and dynamic control of gene expression in bacteria. In addition to riboswitches that can activate gene expression on binding synthetic ligands, we will also apply our strategy to re-engineer riboswitches which can block gene expression in response to selected synthetic molecules. We will also couple multiple switches together, in a tandem arrangement, to enable a more digital control of gene expression. For example we propose to develop a genetic switch which can be turned on with one ligand, activating gene expression, before being switched off with a second distinct ligand. In addition we will also show how the mutually orthogonal riboswitches can be employed to affect the simultaneous and differential control of multiple genes in bacteria. The new genetic switches we develop could be used to study the function of genes in bacteria. In the case of pathogenic bacteria, which cause disease, expression tools based on orthogonal riboswitches could be used to validate new targets for antibiotics that could lead to the development of new antimicrobial treatments. The riboswitch expression tools could also be used to aid production of proteins in bacteria, which could include therapeutically important proteins (biopharmaceuticals) or industrial important enzymes (biocatalysts). In addition, orthogonal riboswitches could be used to control multiple genes which encode metabolic pathways leading to natural product based drugs, biofuels and other commercially important products which are emerging as targets for synthetic biology. During this project we will be working to develop orthogonal riboswitches with the required properties that will allow us to demonstrate their utility in these important applications.