Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase

International audience12 Adjustment of plasmid copy number resulting from the balance between positive and negative impacts 13 of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. 1

Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening

International audience11 Application of droplet-based microfluidics for the screening of microbial libraries is one of the 12 important ongoing development in functional genomics/metagenomics. In this article, we propose a 13 new method that can be employed for the high-throughput profiling of cell growth. It consists in light-14 driven labelling droplets that contain growing cells directly in microfluidics observation chamber, 15 followed by recovery of the labelled cells. This method is based on intracellular expression of green to 16 red switchable fluorescent proteins. The proof of concept is established here for two commonly used 17 biological models, E.coli and S.cerevisiae. Growth of cells in droplets was monitored under a 18 microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was 19 switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then 20 successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. 21 Finally, the application of this method for different kind of screenings, in particular of metagenomic 22 libraries, is discussed and this idea is validated by the analysis of a model mini-library. 2

Genetic Incorporation of Non-canonical Amino Acids in Anti-HER2 VHH: Expression and Characterization

International audienceNanobodies-or VHH-are small proteins (~120 residues) issued from antibodies with intact recognition for the original target of the antibody. In the present study, we show the possibility of incorporating non-canonical amino acids at precise locations of the sequence via classical genetic techniques (Genetic Code Expansion). We demonstrate that the amount of recombinant protein obtained is compatible with a large production format. We show that this protein can be purified, that its sequence corresponds to the theoretical molecular weight, and that the two non-canonical amino acids are incorporated at the desired locations of the sequence. Finally, we show by surface plasmon resonance (SPR) that the affinity of these VHHs is maintained towards their target, HER2