Single-molecule Imaging of the Cell Division Ring in Escherichia coli Using the ALFA-tag

Abstract: The use of super-resolution (SR) microscopy is an important tool for understanding the inside mechanisms of bacterial cells. However, for SR imaging, the labelling of the proteins of interest is a great challenge as flourescent proteins (FPs) are often too big to be directly fused to the target protein and traditional immunolabelling with antibodies creates too long separation between the fluorophore and the target protein. In an attempt to overcome this hurdle, the Escherichia coli (E. coli) cell division protein FtsZ is in this project fused to a nanotag (NT) that is subsequently labelled with a nanobody (NB). The ALFA-tag, a short amino acid peptide, is chromosomally fused to the target protein, creating a MG1655/FtsZ-ALFA strain where all FtsZ proteins have an ALFA-tag attached. Recognising the ALFA-tag is the NB αALFA (anti-ALFA) which is fused to a FP and expressed from a plasmid. The MG1655/FtsZ-ALFA strain is labelled using standard plasmid transformation which allows for live cell imaging of the division ring in E. coli. Both FPs sfGFP and mEos3.2 are used for labelling which means that the cells can be imaged in epifluorescence microscopy and single-molecule Photo-Activated Localisation Microscopy (PALM), and even single-molecule time lapses of the constricting FtsZ-ring is possible. This system is also applicable to other bacterial proteins.