Spatial organisation and mechanical control of gene expression on the bacterial chromosome
Lead Research Organisation:
University of Oxford
Department Name: Biomedical Imaging CDT
Abstract
A crucial condition for life at any level of organisation is the ability to both perceive and correctly respond to a dynamic environment. At the cellular level, complex signalling mechanisms that sense this environment lead ultimately to the regulation of gene expression in the chromosome. The bacterial chromosome is linearly compacted by a factor of approximately 1000 to fit within a region called the nucleoid, supporting gene regulation and correct DNA segregation. Understanding the mechanisms of gene regulation requires the study of the link between chromosome organization and function an open question made challenging by the dynamic nature of the nucleoid.
A common assumption is that the sensing and regulation machinery operates on a purely chemical basis. However, evidence is emerging across both eukaryotes and prokaryotes to suggest that chemistry alone is insufficient to fully explain gene regulation hinting at coupling between biochemical and mechanical processes.
Building upon existing knowledge and expertise in bacterial systems, the impact and objective of our work will be further study of transcription regulation and organisation, and the role of mechanical forces on it.
This work represents a novel collaboration between two Biological Physics research groups in the Department of Physics at the University of Oxford: The Single Molecule "Gene Machines" Group led by Prof. Achillefs Kapanidis, who have extensive expertise in both bacterial transcription and single-molecule super-resolution imaging, both in-vivo and in-vitro.
The Nanoscience for Medicine Group, led by Prof. Sonia Contera, who offer expertise in cell mechanics, and nanoscale mechanical characterisation and manipulation of materials.
Chromosomal organization in normal conditions
In this work, we will develop a novel genetic manipulation system based on mutated CRISPR dCas9, working closely with Dr. Hafez El Sayyed from the "Gene Machines" group - this could be used for both fluorescent tagging and biochemical interrogation. The novel system will enable simultaneous multi-colour fluorescent imaging of genetic loci, and will be used with single-molecule super-resolution microscopy to investigate the structure of the chromosome in 3D space. Specifically we will investigate the relative proximity of operons in wild-type species, and of the remaining number of operons in mutant strains; the potential organisation levels of the nucleoid.
Another level of organization may be provided by the phenomenon of liquid-liquid phase separation, which has recently been implicated in the formation of membrane-less organelles, most interestingly the eukaryotic nucleolus. Naturally, this raises the possibility of the existence of similar mechanism in the bacterial nucleoid - subject to time constraints; this could also investigated using multi-colour fluorescence imaging.
Chromosomal organization under antibiotic-induced stress Imaging chromosomal organization as per Aim 1 also offers the prospect of integrating the work with an ongoing, industry-relevant project on rapid detection of antimicrobial resistance at the single cell level. Here, we will establish antibiotic resistance phenotypes based on changes to nucleoid structure in response to antimicrobial agents, and complement that with species identification from targeted probes - both at the single cell level. Combining that with wide field microscopy offers the prospect of establishing a clinical assay. We will provide proof-of -concept results, working firstly with homogenous samples and moving towards heterogeneous clinical isolates. Collaboration on this initiative is already in place between the Gene Machines group, and the John Radcliffe Hospital in Oxford. Aim 3 The role of mechanical forces in chromosomal organization and regulation
Mechanical forces can affect gene regulation in at least two distinct ways. The first is by triggering of mechano-sensitive biochemical pathways a known t
A common assumption is that the sensing and regulation machinery operates on a purely chemical basis. However, evidence is emerging across both eukaryotes and prokaryotes to suggest that chemistry alone is insufficient to fully explain gene regulation hinting at coupling between biochemical and mechanical processes.
Building upon existing knowledge and expertise in bacterial systems, the impact and objective of our work will be further study of transcription regulation and organisation, and the role of mechanical forces on it.
This work represents a novel collaboration between two Biological Physics research groups in the Department of Physics at the University of Oxford: The Single Molecule "Gene Machines" Group led by Prof. Achillefs Kapanidis, who have extensive expertise in both bacterial transcription and single-molecule super-resolution imaging, both in-vivo and in-vitro.
The Nanoscience for Medicine Group, led by Prof. Sonia Contera, who offer expertise in cell mechanics, and nanoscale mechanical characterisation and manipulation of materials.
Chromosomal organization in normal conditions
In this work, we will develop a novel genetic manipulation system based on mutated CRISPR dCas9, working closely with Dr. Hafez El Sayyed from the "Gene Machines" group - this could be used for both fluorescent tagging and biochemical interrogation. The novel system will enable simultaneous multi-colour fluorescent imaging of genetic loci, and will be used with single-molecule super-resolution microscopy to investigate the structure of the chromosome in 3D space. Specifically we will investigate the relative proximity of operons in wild-type species, and of the remaining number of operons in mutant strains; the potential organisation levels of the nucleoid.
Another level of organization may be provided by the phenomenon of liquid-liquid phase separation, which has recently been implicated in the formation of membrane-less organelles, most interestingly the eukaryotic nucleolus. Naturally, this raises the possibility of the existence of similar mechanism in the bacterial nucleoid - subject to time constraints; this could also investigated using multi-colour fluorescence imaging.
Chromosomal organization under antibiotic-induced stress Imaging chromosomal organization as per Aim 1 also offers the prospect of integrating the work with an ongoing, industry-relevant project on rapid detection of antimicrobial resistance at the single cell level. Here, we will establish antibiotic resistance phenotypes based on changes to nucleoid structure in response to antimicrobial agents, and complement that with species identification from targeted probes - both at the single cell level. Combining that with wide field microscopy offers the prospect of establishing a clinical assay. We will provide proof-of -concept results, working firstly with homogenous samples and moving towards heterogeneous clinical isolates. Collaboration on this initiative is already in place between the Gene Machines group, and the John Radcliffe Hospital in Oxford. Aim 3 The role of mechanical forces in chromosomal organization and regulation
Mechanical forces can affect gene regulation in at least two distinct ways. The first is by triggering of mechano-sensitive biochemical pathways a known t
Planned Impact
The UK has made a significant research impact in the area of biomedical imaging, especially given the size of its research volume. This impact was highlighted in the 2012 EPSRC/MRC Report on Medical Imaging Technologies, that placed the UK first for relative world impact in the neuroimaging field, and third in the world for research in radiology, nuclear medicine and medical imaging (see Appendix 1 of that report). However, the UK does not have a good track record in translating its medical imaging technologies into commercial enterprises. Indeed, most of the major medical imaging technology companies are based outside the UK.
Based on this excellence of biomedical imaging research expertise, however, an opportunity does exist to promote enterprise in the UK, which ultimately may lead to the growth of smaller specialist companies, particularly in the area of supporting drug discovery and assessment of pharmaceutical efficacy. For the pharmaceutical industry the ideal situation is to partner with academically strong medical centres via specialist contract research organizations (of the type represented by one of our industry partners P1Vital) who have imaging experts to guide the complex trials work that is required. In order to prevent emerging markets, with their increasingly competitive academic centres, from being first choice options for hosting such industries, the UK must train a larger pool of entrepreneurially minded imaging scientists.
The other major beneficiary of biomedical imaging science is in the healthcare sector, where NHS delivery costs are rising dramatically, and more focused and quantitative characterization of patients and their treatment progression will be needed. This is true across all scales of imaging, from better tissue characterization at the cellular level (from biopsies and via endoscopic procedures), all the way to human-organ and whole-body imaging methods. The opportunities for cost savings for a more personalized medicine delivery are enormous, but only provided that carefully targeted imaging procedures can be generated and used in combination with personalized genetic information. If successful, imaging could help greatly reduce healthcare costs by better stratifying patients for specific treatments, and by ensuring via longitudinal follow up that those treatments are being effective.
Clearly the biggest impact of the CDT, however, will be the work that the projected 75+ students perform once they complete their studies. This injection of highly trained and inter-connected imaging scientist experts will maintain UK academia's prominence in this field and will greatly strengthen UK industry and the UK healthcare sector. Based on past experience we would expect approximately 60% will move straight into academic research and 20% into industrial research. The remaining students will go into a variety of careers including the healthcare sector and other professional careers. Given the industrial involvement and stimulation in this CDT we would also expect several of our students to be attracted towards an entrepreneurial pathway and to form their own startup companies (e.g. the existing DTCs at the maths/physical/biomedicine interface in Oxford have resulted in 12 such startups). This demonstrates the likely impact of the career development opportunities provided by the ONBI CDT programme, and the resulting excellent employment prospects. Academically we would expect, based on previous and existing similar programmes, that each student will publish 2-3 journal papers arising from their doctoral work, including many in high impact journals, and likely some will file patents. It should finally be noted that all students will be required to participate in public and schools outreach activities in the later years of their training, with the hope and expectation that this will be an activity that they continue beyond their training, thus with a lasting impact.
Based on this excellence of biomedical imaging research expertise, however, an opportunity does exist to promote enterprise in the UK, which ultimately may lead to the growth of smaller specialist companies, particularly in the area of supporting drug discovery and assessment of pharmaceutical efficacy. For the pharmaceutical industry the ideal situation is to partner with academically strong medical centres via specialist contract research organizations (of the type represented by one of our industry partners P1Vital) who have imaging experts to guide the complex trials work that is required. In order to prevent emerging markets, with their increasingly competitive academic centres, from being first choice options for hosting such industries, the UK must train a larger pool of entrepreneurially minded imaging scientists.
The other major beneficiary of biomedical imaging science is in the healthcare sector, where NHS delivery costs are rising dramatically, and more focused and quantitative characterization of patients and their treatment progression will be needed. This is true across all scales of imaging, from better tissue characterization at the cellular level (from biopsies and via endoscopic procedures), all the way to human-organ and whole-body imaging methods. The opportunities for cost savings for a more personalized medicine delivery are enormous, but only provided that carefully targeted imaging procedures can be generated and used in combination with personalized genetic information. If successful, imaging could help greatly reduce healthcare costs by better stratifying patients for specific treatments, and by ensuring via longitudinal follow up that those treatments are being effective.
Clearly the biggest impact of the CDT, however, will be the work that the projected 75+ students perform once they complete their studies. This injection of highly trained and inter-connected imaging scientist experts will maintain UK academia's prominence in this field and will greatly strengthen UK industry and the UK healthcare sector. Based on past experience we would expect approximately 60% will move straight into academic research and 20% into industrial research. The remaining students will go into a variety of careers including the healthcare sector and other professional careers. Given the industrial involvement and stimulation in this CDT we would also expect several of our students to be attracted towards an entrepreneurial pathway and to form their own startup companies (e.g. the existing DTCs at the maths/physical/biomedicine interface in Oxford have resulted in 12 such startups). This demonstrates the likely impact of the career development opportunities provided by the ONBI CDT programme, and the resulting excellent employment prospects. Academically we would expect, based on previous and existing similar programmes, that each student will publish 2-3 journal papers arising from their doctoral work, including many in high impact journals, and likely some will file patents. It should finally be noted that all students will be required to participate in public and schools outreach activities in the later years of their training, with the hope and expectation that this will be an activity that they continue beyond their training, thus with a lasting impact.