Elisabeth Bobkova

@bobez.bsky.social

70 Followers 110 Following

PostDoc at Erb Lab, science policy enthusiast, scientific illustrator, metalhead. Yay to using ML, physics and molecular biology to engineer new synbio systems

Very clean, very empty lab space that I am leaving behind. All in pretty white and with pipettes awaiting their new master
Panel A depicts a typical one-pot modification and peptide diffusion measurement. In brief, the pre-formed vesicles containing LgBit are combined with the MBP-dark breakage control and purified alpha-Hemolysin monomers. The solution is incubated for 30min at RT to allow for pore formation. After that the modification solution (here a fluorescent maleimide conjugate) is added to the mix and everything is incubated at 37°C for 2h to allow maximal modification of the pores post-insertion. The sample is then transferred to well-plates and the luciferase substrate together with the reporter peptide is added. The luminescence measurement is carried out in a platereader. Panel B showcases the chemical structure of the fluorophore coupled to maleimide used for pore modification. Panel C showcases the results of measurements of all 3 pore variants (wild type, 0C and 117C variants) with and without maleimide modification. The signal is normalized to the base reporter (r99), with results depicted to fused r99 constructs - neutrally charged unstructured peptide, and 2 negatively charged helices with different charge distributions. No significant changes in apparent peptide diffusion can be observed for wildtype and 0C pore variants, while a significant change in luminescence is detected between modified and unmodified pore for the 117C variant for negatively charged helices, but not for unstructured neutral reporter construct, suggesting charge and position dependent selectivity in peptide diffusion of the modified pore.
The depiction of the nanoluciferase based assay setup followed by measurements of the breakage contributions. Panel A depicts the setup of the breakage controlled nanoluciferase assay. In brief, the large unilamellar vesicles encapsulate the large bit (LgBit) of the split nanoluciferase, while the substrate, the breakage control (dark peptide fused to MBP) and the reporter peptide (small bit of the nanoluciferase) are added to the outside solution. In case the vesicles are intact, no luminescence signal is generated; in case of vesicle breakage the large bit is inactivated by the MBP-dark breakage control - no luminescence signal is generated; in case of successful pore insertion and reporter peptide diffusion inside the vesicle, a functional nanluciferase is formed, enabling substrate conversion with luminescence signal generation. Panels B and C depict example measurements with and without the addition of a breakage control. The luminescence values taken at t=30min are substantially higher for both, the buffer only and alpha-hemolysin containing system, while the breakage control containing samples show an overall lower signal. Particularly, the signal with alpha-hemolysin containing vesicles shows a 7-fold drop for the breakage-controlled sample compared to measurements without breakage control
ChmeraX visualizations of alpha-Hemolysin in different assembly states. Panel A showcases the monomer of aHL with the cysteine modifications at positions 0 and 117c highlighted in violet and pink respectively. Panel B shows the assembled heptamer from the side. Panel C depicts a top-view on the assembled alpha-Hemolysin pore with peptide-modifications attached to the cysteine at the pore entry (0C) position highlighted in orange. The peptides are predicted (alphafold multimer) to forma mesh at the entry of the pore
Beth standing in front of her Poster with a coffee looking happy and well caffeinated. The poster features her work on chemically tunable permeability of engineered alpha-Hemolysin in synthetic membranes