Seeger, M. | bioRxiv Science

bioRxiv2018

Engineered Peptide Barcodes for In-Depth Analyses of Binding Protein Ensembles

Binding protein generation relies on laborious screening cascades that process candidate molecules individually. To break with this paradigm, we developed NestLink, a binder selection and identification technology able to biophysically characterize thousands of library members at once without handling individual clones at any stage of the process. NestLink builds on genetically fused barcoding peptides, termed flycodes, which are designed for maximal detectability by mass spectrometry and serve as unique molecular identifiers for accurate deep sequencing. We applied NestLink to overcome current limitations of binder generation. Rare binders against an integral membrane protein were identified directly in the cellular environment of a human pathogen. Hundreds of binder candidates were simultaneously ranked according to kinetic parameters. Adverse effects of target immobilization were overcome by selecting nanobodies against an ABC transporter entirely in solution. NestLink may provide a basis for the selection of tailored binder characteristics directly in tissues or in living organisms.

bioengineering↗

bioRxiv2018

Mid-infrared optoacoustic microscopy with label-free chemical contrast in living cells and tissues

We developed mid-infrared optoacoustic microscopy (MiROM), a bond-selective imaging modality that overcomes water/tissue opacity and depth limitations of mid-infrared sensing allowing uncompromised live-cell/thick-tissue mid-infrared microscopy with up to three orders of magnitudehigher sensitivity than other vibrational imaging modalities; such as Raman. We showcase the functional label-free biomolecular imaging capabilities of MiROM by monitoring the spatiotemporal dynamics of lipids and proteins during lipolysis in living adipocytes. Since MiROM, contrary to Ramanmodalities, is not only able to detect lipids and proteins, but also important metabolites such as glucose without the need of labels, here we discuss how MiROM yields novel functional label-free abilities for a broader range of analytical studies in living cells and tissues.

cell biology↗

bioRxiv2017

Degradation of benzene by the heavy-metal resistant bacterium Cupriavidus metallidurans CH34 reveals its catabolic potential for aromatic compounds

Benzene, toluene, ethylbenzene and the three xylene isomers are monoaromatic contaminants widely distributed on polluted sites. Some microorganisms have developed mechanisms to degrade these compounds, but their aerobic and anaerobic degradation is inhibited in presence of heavy metals, such as mercury or lead. In this report, the degradation of benzene and other aromatic compounds catalyzed by the metal resistant bacterium Cupriavidus metallidurans CH34 was characterized. A metabolic reconstruction of aromatic catabolic pathways was performed based on bioinformatics analyses. Functionality of the predicted pathways was confirmed by growing strain CH34 on benzene, toluene, o-xylene, p-cymene, 3-hydroxybenzoate, 4-hydroxybenzoate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, homogentisate, catechol, naphthalene, and 2-aminophenol as sole carbon and energy sources. Benzene catabolic pathway was further characterized. Results showed that firstly benzene is transformed into phenol and, thereafter, into catechol. Benzene is degraded under aerobic conditions via a combined pathway catalyzed by three Bacterial Multicomponent Monooxygenases: a toluene-2-monoxygenase (TomA012345), a toluene-4-monooxygenase (TmoABCDEF) and a phenol-2-hydroxylase (PhyZABCDE). A catechol-2,3-dioxygenase (TomB) expressed at early exponential phase cleaves the catechol ring in meta-position; an ortho-cleavage of catechol is accomplished by a catechol-1,2-dioxygenase (CatA) at late exponential phase instead. This study additionally shows that C. metallidurans CH34 is capable of degrading benzene in presence of heavy metals, such as Hg(II) or Pb(II). This capability of degrading aromatic compounds in presence of heavy metals is rather unusual among environmental bacteria; therefore, C. metallidurans CH34 seems to be a promising candidate for developing novel bioremediation process for multi-contaminated environments.\n\nHIGHLIGHTSO_LIThe strain Cupriavidus metallidurans CH34 is capable to degrade benzene aerobically\nC_LIO_LIBenzene oxydation is mediated by bacterial multicomponent monoxygenases\nC_LIO_LIStrain CH34 is able to grow using a broad range of aromatic compounds as sole carbon and energy source\nC_LIO_LIBenzene degradation occurs even in presence of heavy metals such as mercury and lead\nC_LI

microbiology↗