Development and implementation of metrology standards for mammalian cell-free systems
Lead Research Organisation:
Imperial College London
Department Name: Dept of Medicine
Abstract
To determine the optimum conditions for cell-extract production in HEK293 and CHO cell-lines, I will generate cell-extracts using a variety of conditions (temperature, CO2 %, culturing media, and incubator shaking speed). In addition, I hope to elucidate the contents of the best performing extracts (highest yield of correctly folded proteins, highest activity) to inform our design process. The contents would also be investigated over the course of a reaction, to determine any bottlenecks that reduce batch run time (depleted NTPs, accumulation of inhibitory intermediates, exhausted energy supply). This will build on the work previously conducted by the Freemont lab, to optimise energy consumption in cell-free systemsI will work with automation and analytics experts in the London DNA foundry to determine the best methods for detecting important reaction components. Specifically, I will use the acoustic liquid handling platforms to facilitate high-throughput cell-free expression.
People |
ORCID iD |
| Paul Freemont (Primary Supervisor) | |
| Caoime Canavan (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/R511961/1 | 01/10/2017 | 31/03/2023 | |||
| 2023469 | Studentship | EP/R511961/1 | 01/10/2017 | 30/09/2022 | Caoime Canavan |
| Description | Currently this research is focusing on developing a new cell-free system in order to produce proteins, including biologics such as antibodies. In the context of this work a cell-free systems utilise cellular lysate to produce proteins outside of the constraints of a living system. This system has many benefits over traditional synthesis methods such as being quicker and cheaper, and enabling synthesis of proteins which are otherwise difficult to produce. The first significant outcome of this research is the establishment of a protocol to produce highly active HEK-293 lysates, which to our knowledge has not yet been published. A methods paper detailing this protocol is currently being written. The establishment of this system allows for new research questions to be answered. These include prototyping and modelling of mammalian protein synthesis, screening of constructs prior to large-scale synthesis and rapid synthesis of biologics, such as antibodies. One area we are beginning to investigate is the metabolism of cell-free systems, and how this can be harnessed to extend run times and protein yields. This is being done in collaboration with the London Biofoundry. Additionally, this system can now be used to develop metrology standards to identify key parameter to consistently develop a robust system. This work will be conducted in collobaration with the national measurements standards insititute; the National Physical Laboratory (NPL). Another outcome of this research, which was published in Margarita et al, 2019, is the development of an aptamer to enable real time tracking and quantification of transcription and translation from the same construct. This facilitated modelling of mammalian cell-free systems. Additionally, detailed analytics of the system are being conducted at the London Biofoundry. This data, together with the aptamer data will enable more details models of the mammalian cell-free system to be developed. This outcome has facilitated a new collaboration with the Richard Murray group at the California Institute of Technology, whom have expertise in modelling cell-free systems. |
| Exploitation Route | Development of an in-house, fully optimised mammalian cell-free system will be used for a multitude of applications, including medical and biotechnology. Additionally, this research has developed an alternative method of producing proteins, which is key for many areas of biological research. Therefore this outcome has the potential to be widely used across many disciplines. As previously mentioned, this system will be used to synthesis structural proteins, which are currently synthesised in vivo which involve challenges including difficulties with transfection and low yields in the desired confirmation. Structural studies are necessary for many areas of medicine and healthcare, therefore the development of this cell-free system will be used to improve research in this area. Additionally one area we are focusing on for the remainder of this funding period is the development of antibodies. The application of this ranges from medical, as large antibodies libraries can be screened prior to synthesis, reducing timings and costs of antibody production, to biotechnology as this system can be used as a method of synthesis antibody for laboratories on an individual need basis, rather than outsourcing to a company which can be costly, particularly when niche antibodies are required. |
| Sectors | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |