Our research is focused on developing new ways to engineer and manufacture mammalian and bacterial extracellular vesicles using synthetic biology approaches. Vesiculab’s Vesi-SEC micro columns are a recent and useful addition to our nanoflow cytometry workflow as part of our ongoing bacterial EV engineering project. Usefully, the Vesi-SEC micro columns are reducing sample background FITC signals likely relating to free-GFP and/or fluorescent molecules found within standard bacterial LB media itself (Figure 1). This has improved and simplified our analysis of control and engineered (e.g., GFP-loaded) bacterial vesicles to the benefit of our ongoing project.
Figure 1 Vesi-SEC micro columns reduce background FITC fluorescence and yellow colouration of JM109 Escherichia coli cell conditioned LB media. [A] Representative Images of cell-conditioned LB media from control (empty vector) and engineered JM109 E. coli (periplasmic localised GFP), pre- and post-Vesi-SEC micro column processing. These strains were cultured overnight at 37oC with 200 rpm shaking then diluted to OD600 0.1 in 100 ml fresh LB. Diluted cultures were subsequently cultured at 37oC with 200 rpm shaking for 24 h. Post-culture, samples were centrifuged 3220xg for 15 minutes to pellet the bacterial cells. Supernatants were subsequently dual filtered using a 0.2 µm standard filter followed by a 0.2 µm Tangential Flow Filtration (TFF) column to isolate bacterial vesicles. [B] Representative FITC and scatter plots.
Testimonial and unpublished pilot data provided by Dr Richard Kelwick and Dr Alexander Webb, Imperial College London.
Dr Richard Kelwick
Dr Richard Kelwick is a senior research associate and former Royal Society of Edinburgh Enterprise Fellow with research interests in synthetic biology, cell-free gene expression systems and extracellular vesicle biology.
Dr Alexander Webb
I am currenlty using synthetic biology to build affordable and molecular cell-based biosensors as generic platforms that can be applied to quickly and easily detect parasites via their protease signatures.