RC/GC developing tools for the remote control of genetic circuits

Abstract

With the advent of industrial biotechnology, we can use microbes to produce myriad valuable compounds with a range of useful applications. These may be drugs, flavourings preservatives or many others. With contemporary synthetic biology approaches we are able to bring together enzymes from a variety of different genomic contexts and build pathways that don’t yet exist in nature. This naturally proposes an increase in complexity in pathway design and function, that will require more complex methods to regulate and control. One way to achieve this regulation is using magnetic fields. Physical stimuli like this hold many advantages over the chemical induction methods used contemporarily. Every cell could be reached simultaneously using physical stimuli, promoting metabolic synchronicity. There is also no risk of some cells sequestering chemical inducers, leaving some cells experiencing greater levels of induction, and others none at all. A magnetic stimulus could penetrate an entire culture and stimulate each cell simultaneously and repeatedly. Magnetic nanoparticles when placed in oscillating magnetic fields have their magnetic dipoles forcibly switched to align with oscillations. At high frequency magnetic oscillations, the MNP loses some energy as heat. By conjugating magnetic nanoparticles with temperature sensitive proteins it may be possible to produce stimulus of specific genetic elements with hitherto unseen precision. To achieve this, microbes must be equipped with two things – a magnetic “aerial” module which can receive the magnetic stimulus, and genetically encoded heat-sensitive apparatus that responds to changes in temperature induced by the magnetic field. This thesis has surveyed the potential for Pd nanoparticles to perform the role of a magnetic aerial, and heterologous gene expression with genes from magnetotactic bacterial genomes to produce magnetic material de novo. This strand of work illuminated the potential for a model contrary to how Pd-NPs are made in E. coli. This being that instead of microbial hydrogenases directly nucleating production of a nanoparticle in the active site of the enzyme, hydrogen produced by microbial hydrogenases reacts with soluble Pd(II) precipitating Pd nanoparticles. Work performed in parallel has provided a novel mechanism to produce magnetic material de novo using heterologous expression of the mms6 gene from Magnetospirulum gryphiswaldense. This work has also used contemporary synthetic biology approaches to produce genetic programs that respond to temperature changes, using the genes tlpA, mogR and gmaR, with mogR and gmaR being new vi additions to the pantheon of genetic elements used in synthetic gene circuits. This thesis contains designs, characterisation and optmisation of functional novel genetic circuits which utilise mogR and gmaR in the pMOGMAR-20K plasmid. Using genetic elements characterised in this work in concert, the apparatus to begin producing novel magnetoreceptive mechanisms in E. coli is now available.