Feasibility and Benchmarking of RiboTite gene expression control technology
Lead Research Organisation:
University of Manchester
Department Name: Chemistry
Abstract
We have developed a novel gene expression technology that has applications in the R&D kits market and in the manufacture of biopharmaceuticals. We plan through this current study to benchmark our expression technology against those systems currently available in the marketplace. This study will showcase the key areas of technical superiority of our technology, inform its subsequent positioning in the market place, inform our business planning and act as an enabling primer for private equity investment. The outputs from this grant will lead to partnering collaborations with industry, provide impact for BBSRC funded research, and potentially lead to wealth generation and job creation for the UK.
Publications
Vincent HA
(2014)
Generation of orthogonally selective bacterial riboswitches by targeted mutagenesis and in vivo screening.
in Methods in molecular biology (Clifton, N.J.)
Robinson CJ
(2016)
Rewiring Riboswitches to Create New Genetic Circuits in Bacteria.
in Methods in enzymology
Morra R
(2016)
Dual transcriptional-translational cascade permits cellular level tuneable expression control.
in Nucleic acids research
| Description | Gene expression is the process by which the genetic information encoded in DNA is transcribed into messenger RNA (mRNA) and then translated into proteins. These proteins can function as enzymes, which catalyse the chemical reactions within cells, termed metabolism. Enzymes are of industrial importance because they can catalyse reactions which generate valuable products (pharmaceuticals, agrochemicals and other materials). Other proteins, called antibodies, are produced in cells, and can bind to foreign cells and other target proteins, as part of our bodies immune response to infection by bacteria and viruses etc. Antibodies and other proteins have been developed as biopharmaceuticals for the treatment of a wide range of diseases. The production of proteins, including enzymes and antibodies, on an industrial scale is of considerable commercial importance, but also very challenging. One potential solution to this problem is to use well characterised bacteria such as E. coli as hosts for the expression of genes encoding proteins of interest. Using existing technology it is possible to overexpress genes in E. coli, leading to production of high levels of the protein encoded by that gene. Whilst this works well in some cases, many proteins are insoluble or toxic when produced at high levels. In this project, we have developed orthogonal riboswitches, which enable dose-dependent control of target gene expression in E. coli in response to the addition of a synthetic compound (inducer). To do this we insert a DNA sequence in front of the target gene, which when transcribed forms a very specific RNA structure (riboswitch) at the beginning of the mRNA that blocks translation. This riboswitch is engineered such that when it binds to the non-natural synthetic inducer, a change in structure occurs, which triggers translation of the mRNA to give protein. We have shown that the orthogonal riboswitches that we have developed can provide precise control over gene expression, such that as we increase the concentration of the synthetic ligand we see a dose-dependent increase in gene expression. This means we can control precisely the timing and levels of protein produced in the E. coli cells, which could increase yields of protein and could circumvent issues associated with the production of toxic or insoluble proteins. In addition to using single riboswitches, we have also developed expression systems incorporating two riboswitches, which provide even more precise dual-control over gene expression. As a consequence of this project, a suite of DNA tools (plasmids) and E. coli strains containing orthogonal riboswitches have been created, and these have been transferred, along with the synthetic inducer, into industrial labs for evaluation in larger scale protein production. |
| Exploitation Route | The suite of DNA tools (plasmids) and E. coli strains containing orthogonal riboswitches have been created, and these have been transferred, along with the synthetic inducer, into industrial labs for evaluation. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | New collaborations have been established with companies, including a UK biopharma CMO. Collaboration with Synthace has led to further funding from TSB for the industrial development of orthogonal riboswitches. The training/experience gained from the project has helped with the career development of the researcher-Co-I, who was awarded a BBSRC David Philips Fellowship for related research. The technician employed on the grant has secured a position in industrial biotechnology with Novartis. |
| Title | RIBOSWITCHES |
| Description | The present invention relates to a system comprising a genetic construct a riboswitch operably linked to a regulatory sequence, and a second genetic construct a coding sequence whose expression is capable of being regulated by a gene product of the first construct. Also provided is a genetic construct comprising one or more riboswitches for regulation of gene expression, wherein preferably a spacer sequence is provided downstream of the riboswitch to enhance expression of a coding sequence which is operably linked to a riboswitch. Ligands, kits, methods, host cells and expression systems are also provided. |
| IP Reference | WO2012153142 |
| Protection | Patent application published |
| Year Protection Granted | 2012 |
| Licensed | No |
| Impact | Dixon, N.; Micklefield, J. Riboswitch-regulated transgene expression systems From PCT Int. Appl. (2012), WO 2012153142 A2 20121115. |