Project properties
Title A toolkit to optimize metabolic pathways in chromosomal free cells
Group Systems and Synthetic Biology
Project type thesis
Credits 36
Supervisor(s) Prof. Maria Suarez Diez, Dr. Stefan Hoffman, Jelmar de Vries
Examiner(s) Prof. Maria Suarez Diez; Dr. Stefan Hoffman
Contact info robert1.smith@wur.nl
Begin date 2024/12/04
End date
Description Chromosome-free cells are an interesting platform for metabolic engineering(1). Since they cannot replicate, they are by nature bio-contained and cannot waste substrate on biomass production. To efficiently convert substrate into product, the enzymes required to perform the desired reactions need to be expressed. Potential roadblocks are the expression levels of the required enzymes and the simultaneous expression of the native metabolism and a synthetic metabolism.
This project aims to develop a workflow in which the regulation of a metabolic pathway can be first optimized with inducible promoters and then be expressed by constitutive promoters dependent on an orthogonal sigma factor. A sigma factor is part of the RNA polymerase holoenzyme and enables transcription from a specific set of cognate promoters. Characterizing a set of inducible promoters(2) and a set of promoters expressed by an orthogonal sigma factor(3) is required for this approach.
The project will involve cloning of a promoter library using a modular cloning toolkit leveraging lab automation. The promoter library will then be characterized for expression strength. Finally, a pathway can be optimized in chromosome-free bacteria.

References

1. Fan C, Davison PA, Habgood R, et al. Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology. Proc Natl Acad Sci. 2020;117(12):6752-6761. doi:10.1073/pnas.1918859117

2. Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat Chem Biol. 2019;15(2):196-204. doi:10.1038/s41589-018-0168-3

3. Bervoets I, Van Brempt M, Van Nerom K, et al. A sigma factor toolbox for orthogonal gene expression in Escherichia coli. Nucleic Acids Res. 2018;46(4):2133-2144. doi:10.1093/nar/gky010



Used skills • Use of lab automation platforms
• Modular Golden Gate cloning (MoClo toolkit)
• Advanced synthetic biology tools
• Metabolic engineering of chromosome-free E. coli
Requirements • Basic microbial cultivation techniques
• General knowledge of molecular microbiology
• Basics of molecular cloning
Ideally having followed one or more of the following courses: Applied Molecular Microbiology (MIB30806), Research Methods Microbiology (MIB30303), Practical Biological Chemistry (BIC10306).