Orthogonal riboswitches as tools for controlling gene expression in bacteria

基因表达是DNA序列信息首先被转录成信使RNA (mRNA)，然后被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）在响应环境条件和其他信号时合成的时间和水平。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，在细菌中分布尤其广泛。通常，这些开关调节特定代谢物的产生、降解或运输。例如，添加a -核糖开关与腺嘌呤结合，激活编码腺嘌呤脱氨酶的mRNA的翻译，该酶可降解腺嘌呤。通过这种反馈机制，细菌可以控制腺嘌呤的细胞水平，腺嘌呤是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）添加了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，我们的目标是通过进一步的遗传操作和开发新的和更有效的合成配体来开发新的正交核开关，这将允许更精确和动态地控制细菌中的基因表达。除了可以激活结合合成配体上的基因表达的核糖开关外，我们还将应用我们的策略重新设计可以阻断基因表达以响应选定的合成分子的核糖开关。我们还将把多个开关耦合在一起，以串联的方式，使基因表达的数字化控制成为可能。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，然后用另一种不同的配体关闭。此外，我们还将展示如何利用相互正交的核开关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。就致病细菌而言，基于正交核糖体开关的表达工具可用于验证抗生素的新靶点，从而可能导致新的抗菌治疗方法的开发。核糖开关表达工具还可以用于帮助细菌产生蛋白质，包括治疗上重要的蛋白质（生物制药）或工业上重要的酶（生物催化剂）。此外，正交核开关可用于控制多种基因，这些基因编码代谢途径，从而产生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目标。在这个项目中，我们将致力于开发具有所需属性的正交核开关，这将使我们能够展示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.