Filip Ilievski defends thesis on a new protein labelling technology for single-molecule studies in bacteria

We congratulates Filip Ilievski on the successful defense of his PhD thesis: "Fluorescence labelling in re-coded E. coli with non-canonical chemical entities. Single-codon labelling for single-molecule tracking." The defense took place at Uppsala University on 17 March 2026, making Filip the third PhD student from UAC's second cohort to complete his doctoral studies. Filip carried out his work under the supervision of Magnus Johansson and focused on developing a new technology to study protein biology in bacteria.

Watching individual proteins inside living cells

Most of what we know about how bacterial proteins work comes from test-tube experiments where proteins are isolated, purified, and studied outside the cell. This experimental setup can be really helpful, but it misses something fundamental: proteins don't work in isolation. They compete, interact, and respond to a dynamic environment that is difficult to replicate in a test tube.

Filip's research takes a different approach, developing tools to watch them where they are naturally found. The central challenge was labelling: to track a single protein molecule under the microscope, you need to attach a fluorescent marker to it, but most available and used markers are too large and interfere with what the protein does.

The method he built is called FLORENCE (Fluorescence Labelling in Re-coded E. coli with Non-canonical Chemical Entities). Instead of fusing a bulky protein tag, FLORENCE installs a non-natural amino acid at one specific position in the target protein. That amino acid carries a small chemical handle, which reacts with a fluorescent dye in a second step. The result is a labelled protein with minimal structural disruption and tracked, one molecule at a time, inside a living bacterial cell.

Three papers making one integrated platform

Filip’s thesis is built on three research papers following a natural workflow through method development and practical applications:

Developing and validating FLORENCE. Filip optimized every component of the system: the enzymatic machinery for incorporating non-natural amino acids, the bacterial strains, the plasmid architecture, and the click chemistry reaction used to attach the tracking dye. A key result was having achieved labelling inside live cells within 30 minutes. Validation with two reporter proteins, one cytoplasmic and one membrane-associated, confirmed that FLORENCE produces single-molecule tracking results comparable to the widely used HaloTag method.

Tracking a previously inaccessible protein. FLORENCE was applied to study elongation factor G (EF-G), an essential motor protein that drives the ribosome forward during protein synthesis. Conventional HaloTag labelling completely disrupted EF-G function and the tagged protein failed to bind ribosomes, rendering the traditional technique unusable. FLORENCE labelling at a single internal position restored ribosome binding and captured EF-G dynamics for the first time in living bacterial cells.

Improving the platform through adaptive evolution. The bacterial strain used in FLORENCE was further optimized through adaptive laboratory evolution. The resulting strain, GRE*, grows closer to wild-type rates under imaging conditions, a practical bottleneck in earlier work. The paper also applies FLORENCE to track EF-Tu, a second elongation factor that, like EF-G, cannot be labelled with conventional approaches.

Why this matters for AMR

Elongation factors, and other protein synthesis components, are targets for clinically used antibiotics, including aminoglycosides. Understanding how these proteins behave inside living cells, how long they bind the ribosome, or how mutations alter their dynamics, is directly relevant to antibiotic mechanisms and resistance. FLORENCE makes this level of observation possible for proteins that were previously inaccessible to single-molecule methods.

More broadly, Filip's work establishes a platform that can be applied to other proteins where bulky tags get in the way. In his thesis, he points to future directions including multi-colour labelling, in-cell FRET experiments between interacting translation factors, and single-molecule studies of initiation, termination, and ribosome recycling, steps in bacterial protein synthesis where direct in vivo kinetic data are still largely missing.

We warmly congratulate Filip on a significant contribution to molecular biology and protein synthesis research. His thesis reflects the depth and ambition of UAC's second PhD cohort, and we look forward to following the next steps in his scientific career.