From carbon nanotubes to zinc porphyrins: engineering proteins to interface with non- biological molecular systems.

细菌对抗生素的耐药性是现代医疗保健中最重要的危机之一。最广泛使用的抗生素（和一般治疗剂）是β-内酰胺类，包括氨苄西林、阿莫西林和甲氧西林。到目前为止，细菌用于赋予β-内酰胺抗性的主要机制是产生β-内酰胺酶，所述β-内酰胺酶水解β-内酰胺环中对其抗菌活性至关重要的假肽键。该项目的目的是产生一种高信息含量的传感器，能够快速检测β-内酰胺酶的存在和类型，以更有效地临床使用当前的β-内酰胺。为了实现这种工程化的β-内酰胺酶抑制蛋白（BLIP）将选择性地与石墨烯场效应晶体管连接，使得蛋白质-蛋白质相互作用通过体结合效应和静电表面相互作用转换为石墨烯桥接微电极的电阻变化。该系统具有感知与单个蛋白质分子相关的事件的潜力。BLIP是理想的传感器，因为它结合各种临床相关的β-内酰胺酶，具有非常独特的表面静电和相互作用特性，这将可能提供离散的电子签名。一个关键的要求是定义和指导BLIP与石墨烯的接口，以最大限度地提高两个组件之间的通信。这将使用合成生物学方法来实现，以在计算机上设计用于重组并入非天然化学手柄（例如苯基叠氮化物化学）的残基位置，这将允许蛋白质与石墨烯的高精度和最佳界面。蛋白质-石墨烯界面的详细单分子分析（AFM/STM）将导致蛋白质-石墨烯-电极生物传感平台的微制造，并通过电导率的变化开始监测和评估结合事件。第一轮项目（琼斯，卡迪夫）。学生将启动第一轮BLIP变体设计，引入选定的突变，并使用重新编程的遗传密码方法，在定义的位置使用非天然氨基酸引入新的反应性手柄。学生将产生并功能测试所选的BLIP变体，以确定突变的影响。第二轮项目（卡迪夫和埃克塞特物理）。学生将开始优化方法，直接组装石墨烯上的BLIP变体。采用两种方法：（1）通过光生反应性氮烯自由基的直接共价键合;（2）？-将加合物堆积到BLIP上。学生将使用单分子成像方法（AFM-STM）分析BLIP-石墨烯界面。功能研究也将通过成像石墨烯上BLIP-β-内酰胺酶复合物的形成开始。

Bacterial resistance to antibiotics is one of the most significant crises in modern healthcare. The most widely utilised class of antibiotics (and therapeutics in general)are the beta-lactams, which include ampicillin, amoxicillin and methicillin. By far the main mechanism bacteria use to confer beta-lactam resistance is the production of beta-lactamases that hydrolyses the pseudo-peptide bond in the beta-lactam ring critical for its antibacterial activity. The aim of this project is to generate a high-information content miniaturised sensor with the capacity to rapidly detect the presence and type of beta-lactamase to allow more effective clinical use of current beta-lactams. To achieve this engineered beta-lactamase inhibitory protein (BLIP) will be selectively interfaced with a graphene field-effect transistor so that protein-protein interactions are transduced into a change in resistance of graphene-bridged microelectrodes through both bulk binding effects and electrostatic surface interactions. This system has the potential to sense events connected to single protein molecules. BLIP is the ideal sensor as it binds a wide variety of clinically relevant beta-lactamases with very distinct surface-facing electrostatics and interaction properties, which will potentially offer discrete electronic signatures. A key requirement is defined and directed interfacing of BLIP with graphene to maximise communication between the two components. This will be achieved using a synthetic biology approach to design in silico residue positions for the recombinant incorporation of non-native chemical handles (e.g. phenyl azide chemistry) that will allow high precision and optimal interfacing of protein with graphene. Detailed single molecule analysis (AFM/STM) of the protein-graphene interface will lead to the microfabrication of the protein-graphene-electrode biosensing platform and begin monitoring and assessing binding events through changes in conductivity. First rotation project (Jones, Cardiff). The student will initiate the first round of BLIP variant design in silico, introduce the selected mutations, and using a reprogrammed genetic code approach incorporate the new reactive handles using non-natural amino acid at the defined positions. The student will produce and functionally test the selected BLIP variants to ascertain the effect of the mutations. Second rotation project (Cardiff and Exeter Physics). The student will begin optimising approaches for directly assembling the BLIP variants on graphene. Two approaches will be taken: (1) direct covalent linkage through the photogeneration of reactive nitrene radicals; (2) Click addition of ?-stacking adducts to the BLIP. The student will analyse the BLIP-graphene interface using single molecule imaging approaches (AFM-STM). Functional studies will also begin through imaging the formation of BLIP-beta-lactamase complexes on grapheme.