19-BBSRC-NSF/BIO Characterizing efficiency and limitations of RNA regulators to achieve robust dynamic behaviours
Lead Research Organisation:
Imperial College London
Department Name: Chemical Engineering
Abstract
Synthetic biology has made it possible to use cells to operate as microscale factories, energy sources, and even computers. However, the introduction of DNA circuits into cells can cause the cells to be stressed, something named as burden. Circuits that are poorly tunable, and non-native to the host often causes undesired cross-interactions and unpredictable responses, and reliable engineering of cells remains a challenge. In particular, the expression of exogenous pathways triggers physiological changes in the host, usually leading to decreased growth due to consumption of cellular resources, a phenomenon known as cellular burden.
These challenges could be solved using RNA-based regulators of gene expression. These are systems which require transcription to occur but not translation, as no protein needs to be produced. The RNA acts as a regulatory molecule that can impact on the behaviour of the circuit in the cells.
RNA circuits are programmable regulatory platform that imposes a lower burden on the host.
Yet, little attention has been dedicated to systematically tuning the behaviour of RNA regulators. This project aims at screening and tuning two classes of RNA regulators. The regulators will then be employed as rapid, tunable components to better understand burden in bacteria.
With improved knowledge of burden, we will modify bacteria to achieve better bioproduction of molecules of interest. Mathematical modelling will help our work by assisting the choice of experiments and circuits to be adopted.
These challenges could be solved using RNA-based regulators of gene expression. These are systems which require transcription to occur but not translation, as no protein needs to be produced. The RNA acts as a regulatory molecule that can impact on the behaviour of the circuit in the cells.
RNA circuits are programmable regulatory platform that imposes a lower burden on the host.
Yet, little attention has been dedicated to systematically tuning the behaviour of RNA regulators. This project aims at screening and tuning two classes of RNA regulators. The regulators will then be employed as rapid, tunable components to better understand burden in bacteria.
With improved knowledge of burden, we will modify bacteria to achieve better bioproduction of molecules of interest. Mathematical modelling will help our work by assisting the choice of experiments and circuits to be adopted.
Technical Summary
Synthetic biology has made it possible to repurpose cells to operate as microscale factories, energy sources, and even computers. However, the introduction of pathways that are poorly tunable, and non-native to the host often causes undesired cross-interactions and unpredictable responses, and reliable engineering of cells remains a challenge. In particular, the expression of exogenous pathways triggers physiological changes in the host, usually leading to decreased growth due to consumption of cellular resources, a phenomenon known as cellular burden. These challenges could be mitigated using RNA-based regulators of gene expression, which are a programmable regulatory platform that imposes a lower burden on the host. Yet, little attention has been dedicated to systematically tuning the response function of RNA regulators. This project aims at screening and tuning the response function of two classes of RNA regulators. The regulators will then be employed as rapid, tunable components to better understand the burden response in E. coli.
With improved knowledge of burden, we will engineer more robust bioproduction in E. coli. At every stage, experiments will be supported and guided by mathematical modelling.
With improved knowledge of burden, we will engineer more robust bioproduction in E. coli. At every stage, experiments will be supported and guided by mathematical modelling.
Publications
Grob A
(2021)
Experimental tools to reduce the burden of bacterial synthetic biology
in Current Opinion in Systems Biology
Grob A
(2024)
Mammalian cell growth characterisation by a non-invasive plate reader assay
in Nature Communications
| Description | Tracking and measuring of RNA regulators and RNA production in live cells is important to understand the dynamics of many cellular regulation pathways and strengthen our ability to exploit these for the development of synthetic biology control tools. One example is the cellular burden response to heterologous expression in bacteria. Dynamic regulation of burden would be useful to characterise to further improve tools to mitigate burden in these cells. Fluorescent RNA aptamers have proven to be useful tools for tracking and visualization of RNA production. However, they can only be adopted downstream of strong viral promoters (i.e. T7,T5) as their signal intensity is too low when natural promoters are adopted. This greatly limits our ability to easily investigate the dynamics of native RNA expression. Recently, novel and stronger aptamers were published, among these two novel broccoli aptamers and the pepper aptamer. These have so far been adopted in in vitro systems and mammalian cells, but not in bacteria. The PDRA Alice Grob started by characterising these three aptamers in E. coli cells and benchmark their features against the standard broccoli aptamer already adopted in bacteria. She characterised their fluorescence signal when they are placed downstream of different promoters, comparing expression from the stronger T7 promoter and native promoters (e.g. wild type and stronger pBad, Lux) to investigate if these aptamers can allow in vivo RNA tracking in bacteria with greater precision. She first compared expression at a define time point and we then show the behaviour over time when aptamers are followed over a 5-hour window. She did this by plate reader and flow cytometry assays in order to provide information on the best protocol to adopt. We want to also compare expression in bacteria to expression in mammalian cells and in vitro, in order to provide a comprehensive understanding of the features and limitations of these aptamers for synthetic biology application. Finally, in collaboration with the UCLA group of Prof Elisa Franco, we are designing a toehold switch based on the pepper aptamer. Initial work done by the PDRA at the beginning of the project showed that these fluorescent aptamers may be too weak to be placed directly downstream of burden responsive promoters in E. coli, which are quite weak promoters. While we continue the characterisation, we are thus designing a toehold switch where pepper is off unless a small trigger RNA binds and activates fluorescence. This would allow us to use the burden responsive promoter to trigger expression of the trigger RNA so to track its production by a toehold-mediated activation. We will also add variants where native small RNAs whose production is linked to cellular stress can be detected. Initial prototypes of this toehold-based switch did not prove functional in bacteria at Imperial College, while they displayed good functionality in in vitro at UCLA. We attributed this to a different stem loop present in the pepper system compared to the published system which is working in our hands. We are thus now working with the UCLA group to modify the toehold stem so as to test the functionality of the new system. |
| Exploitation Route | It is still quite early to say as the award is still ongoing. Our characterisation of fluorescent aptamers can prove really useful in the field to provide quantitative information on the features of novel aptamers not characterised yet in bacteria and how this compares to performance in synthetic and mammalian cells. If it works, the toehold switch we are designing could prove very useful indeed to allow tracking and analysis of RNA production there where normal native promoters may be too weak to be directly detected with techniques other than RNAseq or qPCR which are more expensive and limited by time point measurements. |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | UCLA collaborations |
| Organisation | University of California, Los Angeles (UCLA) |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | We are actively collaboration with Prof. Elisa Franco on the project funded by the award. We are working on building synthetic constructs that express the fluorescent RNA aptamers pepper and corn under the control of pBAD promoters (weaker and stronger). We are setting up protocols to dynamically track fluorescence from the aptamers to establish if it is possible to use them as tool for tracking the bacterial burden response. The group is contributing with experimental expertise not present in the collaborators lab, especially in vivo work in bacteria. We are also contributing ideas (dynamic tracking), plasmid constructs and burden responsive constructs that were developed in our group. The PDRA has been trained on plate reader and flow cytometry equipment usage and molecular cloning in E. coli. |
| Collaborator Contribution | Prof Franco is in charge of the modelling side of the project and in vitro work on constructs. She is currently characterising in vitro toe-hold mediated sensing using novel constructs developed in her lab that can be employed in my lab for sensing of burden and dynamic tracking of RNA aptamer activity. |
| Impact | No outputs yet |
| Start Year | 2021 |