| Popis: |
In recent years, nanopores have become an important tool in biotechnology especially in biosensing and DNA sequencing. The capacity to engineer nanopores with tailored properties and functions is key to their biotechnological development and provides new fundamental insight into their mechanisms of action. To date, most construction efforts have been realised with rational, structure-guided approaches while genetic screening systems are not widely used. Addressing this technological gap, a genetic reporter assay was developed based on genetically-encoded fluorescent protein sensors to enable a highresolution, quantitative read out of nanopore function in E. coli. The capacity of this assay was demonstrated by dissecting molecular features and functions of the bacteriophage pinholin S21-68 in highresolution genetic screens on different platforms and complemented with high-resolution electrical recordings. This class of membrane peptides is part of the bacteriophage life cycle and very efficient in cell lysis initiation. Detailed analysis of the first transmembrane domain revealed key functions of single amino acids with positively charged residues as main regulators of nanopore stabilization. In addition, several different nanopores were tested with this system and demonstrated the expandability of the screen for further application. |
