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Directed Evolution in vivo enabled through genetic.. (DESB)
Directed Evolution in vivo enabled through genetic circuits in a Synthetic Biology approach
(DESB)
Date du début: 1 juin 2014,
Date de fin: 31 mai 2016
PROJET
TERMINÉ
Given our limited ability to rationally design proteins to our needs, their supply for biotechnological solutions leading to a sustainable Bioeconomy is relying on proteins found in nature and methods for deriving improved versions. Proteins evolved their structure to fulfil certain functions in very specific niches, but these do not resemble optimal properties in the new context of biotechnological applications. Hence, improving the activity, affinity and stability of a protein is key, but technologically challenging. The current methods for improving proteins through directed evolution require intense human intervention and are limited in their ability to sample sequence space beyond single and double mutations. This project will establish a novel system, in which the whole process of mutation and selection takes place in vivo inside an E. coli host, and can be controlled through a genetic network designed using a Synthetic Biology approach. It will allow for the exploration of a larger sequence space during the evolutionary process, while controlling mutagenesis rate and selection stringency. Our preliminary data demonstrates a groundbreaking novel method to target mutagenesis in vivo. I will extend its mutation diversity by recruiting engineered error prone polymerases to the site of the targeted DNA damage. Using a SynBio approach, I will develop a control circuitry, which will stop the targeted mutagenesis activity, once a variant fulfils the selection criteria. While protecting the identity of the selected sequence, it will mark the successful cell by expression of a fluorescent reporter for selection. In a prototype application I will generate variants of the Quorum Sensing transcription factor, LuxR, with high affinities to different signalling molecules – a potential toolset for orthogonal cellular signalling applications in industrially relevant co-cultures. The resulting directed evolution system will be readily adaptable to different proteins.
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