Re-engineering of CUP1 promoter and Cup2/Ace1 transactivator to convert Saccharomyces cerevisiae into a whole-cell eukaryotic biosensor capable of detecting 10 nM of bioavailable copper

Žunar, Bojan; Mosrin, Christine; Bénédetti, Hélène; Vallée, Béatrice

doi:10.1016/j.bios.2022.114502

Cited by 8 publications

(11 citation statements)

References 81 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, the diffusion of exogenous copper ions into the plug would raise the ymNeongreen/ymScarletI ratio within the cells. We embedded these cells in hydrogel plugs and incubated them at 30 °C/20 h in water or media, without Cu 2+ or with 10 µM Cu 2+ , a concentration easily detected by our copper biosensor [ 36 ], yet biocompatible, i.e. non-toxic for yeast cells.…”

Section: Resultsmentioning

confidence: 99%

“…Finally, in larger microcolonies, outer layers emitted the strongest signal, probably because their cells were in exponential phase and continuing to responding to Cu 2+ . Thus, although hindered by hydrogel, Cu 2+ could diffuse into the plugs, where it remained bioavailable [ 36 ] and readily detectable.…”

Section: Resultsmentioning

confidence: 99%

“…Copper was supplemented as CuSO 4 (Merck, Germany). Strains expressing ymNeongreen [ 38 ], QUEEN-2 m [ 39 ], and sfpHlourin [ 40 ] were grown in -his medium, while strain expressing copper biosensor [ 36 ] was grown in -ura medium.…”

Section: Methodsmentioning

confidence: 99%

“…Correct targeting and multiple integration were verified by colony PCR with NEB OneTaq MasterMix. The BY 4742 strain carrying chromosome-integrated pCu2 was obtained from Žunar et al [ 36 ].…”

Section: Methodsmentioning

confidence: 99%

“…One example would be a classical whole-cell biosensor that reacts to an analyte of interest by inducing transcription of the reporter gene while constitutively expressing an additional reporter to normalize the induced signal. We have recently developed one such biosensor to quantify bioavailable copper [ 36 ], a ubiquitous and essential heavy metal that is toxic at high concentrations and whose environmental presence thus needs to be strictly monitored [ 37 ]. Of course, the successful application of this construct in the hydrogel-embedded cells would demonstrate that Pluronic F127-based ELMs can be used as easily deployable environmental biosensors.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Confocal imaging of biomarkers at a single-cell resolution: quantifying 'living' in 3D-printable engineered living material based on Pluronic F-127 and yeast Saccharomyces cerevisiae

et al. 2022

Self Cite

View full text Add to dashboard Cite

Background Engineered living materials (ELMs) combine living cells with non-living scaffolds to obtain life-like characteristics, such as biosensing, growth, and self-repair. Some ELMs can be 3D-printed and are called bioinks, and their scaffolds are mostly hydrogel-based. One such scaffold is polymer Pluronic F127, a liquid at 4 °C but a biocompatible hydrogel at room temperature. In such thermally-reversible hydrogel, the microorganism-hydrogel interactions remain uncharacterized, making truly durable 3D-bioprinted ELMs elusive. Methods We demonstrate the methodology to assess cell-scaffold interactions by characterizing intact alive yeast cells in cross-linked F127-based hydrogels, using genetically encoded ratiometric biosensors to measure intracellular ATP and cytosolic pH at a single-cell level through confocal imaging. Results When embedded in hydrogel, cells were ATP-rich, in exponential or stationary phase, and assembled into microcolonies, which sometimes merged into larger superstructures. The hydrogels supported (micro)aerobic conditions and induced a nutrient gradient that limited microcolony size. External compounds could diffuse at least 2.7 mm into the hydrogels, although for optimal yeast growth bioprinted structures should be thinner than 0.6 mm. Moreover, the hydrogels could carry whole-cell copper biosensors, shielding them from contaminations and providing them with nutrients. Conclusions F127-based hydrogels are promising scaffolds for 3D-bioprinted ELMs, supporting a heterogeneous cell population primarily shaped by nutrient availability. Graphical Abstract

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%