Student

Federica Raguseo

Title

DNA phase-separation as a general mean of regulating gene expression in hybrid cells

Supervisors

• Marco Di Antonio
• Lorenzo Di Michele

Abstract

Liquid-liquid phase separation is increasingly recognised as a key mechanism to regulate gene expression in living cells by controlling the accessibility of genetic material and its co-localisation with transcription machinery. Although mechanisms that trigger phase separation in biological cells are not fully understood, a similar control of transcriptional activation could be recapitulated in hybrid cells, engineered from the bottom-up by combining biological and man-made components. Programming the condensation of genetic material (DNA) and its co-localisation with cellular machinery is a particularly promising approach to regulate transcription. DNA aggregation can be induced by wellunderstood canonical and non-canonical secondary structures, and its occurrence can thus be controlled by a variety of physical stimuli, including temperature, crowding agents and exposure to certain ions. Herein, we propose to generate a hybrid cell system that can be transcriptionally activated by triggering nucleic-acid phase separation, offering an orthogonal solution to current methods that fully rely on small-molecule treatment (e.g. IPTG) and cannot be controlled easily by physical means. Besides offering an alternative method to regulate gene-expression in artificial cells, this project will yield insights on the fundamental physical processes that might be responsible of gene-regulation via liquid-liquid phase separation in living cells

Student

Aileen Cooney

Title

Engineering motile artificial cells capable of swimming up concentration gradients

Supervisors

• Lorenzo Di Michele
• Yuval Elani
• Pietro Cicuta

Abstract

Billions of years of evolution shaped cells into highly sophisticated micromachines, whose intricate network of molecular interactions are difficult to unravel. Bottom-up synthetic biology aims at constructing artificial cells by combining a small number of molecular agents into compartmentalised microenvironments. This reductionist approach enables the study of biological processes in a simplified setting. More importantly however, it offers the opportunity of producing smart cell-like agents to tackle pressing needs in diagnostics, therapeutics, biosynthesis, and bioremediation. Most attempts at engineering life-like 'behaviours' into artificial cells have focused on metabolism, energy generation, computation, and communication. One behaviour which has been neglected so far is motility: directed motion towards a target site. Motility (e.g. swimming and crawling) is a characteristic that is found across all life classes: from unicellular photosynthetic organisms that perform vertical migrations to optimise light exposure, swimming sperm cells, and macrophages that chase down pathogens. In most instances, cells are able to direct their motion following environmental cues, typically gradients in light intensity (phototaxis), temperature (thermotaxis), or the concentration of chemicals (chemotaxis). Despite the unquestionable benefits that controllable taxis would bring for most foreseen applications of artificial cells, viable technologies for engineering motion in synthetic cells have not been developed. This is because the protein assemblies needed to drive motility (e.g. flagella) are the most complex macromolecular structures in existence, and reconstituting them into synthetic systems is simply not possible using current state of the art. In this project we will develop a cellular bionics solution to this challenge: instead of using native cellular machineries, we will develop novel nanotechnologies based on DNA biophysics to propel a synthetic cell forwards, up a concentration gradient, with cell engineered to elicit a response (protein synthesis) when reaching its target site.

Student

Gabriela Sachet-Fernandez

Title

Unlocking a new toolkit for mediating protein-based interactions between synthetic cells and real cells

Supervisors

• Rudiger Woscholski
• Oscar Ces

Abstract

A major bottleneck in the field of cellular bionics and bottom-up synthetic biology is the ability to reversibly decorate the chassis of synthetic cells with user defined proteins. Unlocking this technology bottleneck would transform our ability to mediate interactions between synthetic cells and between synthetic cells and real cells across extended length scales including tissues based materials. This project will lead to the development of a new generation of artificial lipids that are able to reversibly bind an extended library of commercially available his-tagged proteins thereby revolutionising our ability to functionalise cellular bionic systems. We will validate this strategy by using this approach to modulate communication between synthetic cells and real cells at the single cell level. In addition by making use of extracellular proteins, such as integrin and cadherin we will manufacture hybrid tissues that accommodate living and synthetic cells. This approach is highly flexible and can be extended to accommodate most commercially available tags.

Student

Lino Prados Martin

Title

Bionic Robotics: Autonomous Biohybrid Machines powered by skeletal muscle tissues

Supervisors

• Molly Stevens
• Mirko Kovac
• Martin Heeney

Abstract

The inspiration from this project lies in the ability of living organisms to interact with and adapt to the changing environment in real time. The Bionic Robotics seeks to develop machines using biologicallyinspired functionality, to create new designs that can interact more effectively with the natural environment. Conventional robotics use rigid components powered by hard actuation techniques, such as hydraulic and electromagnetic systems. These well-developed robotic systems have wide applications in industry, however suffer from poor autonomy, low adaptability to dynamic environments, and low scalability. Bionic robotics, inspired by living organisms, aim to endow existing robots with sensing and response capabilities to allow them to autonomously interact with unstructured environments. This project will develop an autonomous soft robotic system. It is powered by biohybrid actuators, and capable of both monitoring its biomolecular environment and responding through changes in motion.

Living components will be integrated into the robot in two fundamental areas: living-cell actuation and bacteria-based biosensing. These two components will be linked using electronic control circuitry. Specifically, this project aims to:

• Create a new biohybrid actuator based on skeletal muscle cells: living cell-based actuation uses the intrinsic motion of cells from contractile tissues to generate motion. It uses molecular motors hierarchically organised to form macroscopic artificial contractile tissues. Skeletal muscle tissue is an attractive candidate for the construction of biohybrid actuators. It can be engineered from the millimetre to meter length and be readily controlled using external stimulation. The student will investigate how these living systems operate and how they can be efficiently used in bionic robotics. Skeletal muscle can be controlled either by electricity or light. Electrical stimulation will be used for initial development of a system. However, this has been shown to induce the degradation of the skeletal muscle tissues over time, hence an optical stimulation approach (via optogenetics) will also be investigated.
  
  
• Build a biosensing interface: living cell-based robots are currently limited by their ability to communicate and respond to complex microenvironments. Creating new communication bridges between machine and surroundings are essential. The student will explore the use of the bacteria Escherichia coli (E.coli) for demonstration to enable communication between the external environment and robotic controlling system. We will choose Isopropyl beta-D-1thiogalactopyranoside (IPTG) as a common chemical inducer. With the presence of IPTG, genetically modified E.coli can express a green fluorescent protein (GFP), which will be detected by the following electronic system.
  
  
• Develop an electronic control system to facilitate communication between actuator and biosensor: the student will develop an electronic interface based on off-the-shelf components to exchange information between the biosensor and cell-actuator. Light-emitting diodes and photodetectors will be used to excite and detect the fluorescence change from the biosensor which will be interpreted by an embedded microprocessor and used to stimulate the livingcell actuator through light pulses

Student

Sabrina Pia Nuccio

Title

Unravelling epigenetic drivers of chemo-resistance by optical editing of DNA-methylation in cells

Supervisors

• Marco Di Antonio
• Robert Brown
• Lorenzo Di Michele

Abstract

Aberrant DNA-methylation is a well-established driver of acquired resistance in ovarian cancer, although such epigenomic changes occur within a high background of passenger events and current epigenetic chemical biology tools generally affect the entire epigenome rather than locus specific. This makes it challenging with current biological tools to investigate the role of key locus specific methylation events in resistance mechanisms. To overcome this, epigenetic editing using CRISPRCas9 fused to epigenetic modifiers is increasingly being evaluated, although current approaches have the limitation of not being inducible. To identify key drivers of resistance and demonstrate phenotypic effects, we aim to generate novel chemical ligands that enable light-controlled positioning of DNA methylation writer and eraser (e.g. DNMTs and TETs) at specific genomic loci by means of CRISPRCas9. These chemical tools will disentangle the role of site-specific methylation in patient-derived cell-lines and xenografts. The light-controlled positioning of the epigenetic modifiers will enable temporal control of epigenetic modification, allowing evolution of resistance and its prevention or resensitisation to be studied in a time-dependent manner that is essential to study dynamic epigeneticbased processes such as acquired resistance to chemotherapy.