Microscopic observation of HeLa cells infected with BactoDHL confirmed the functionality of the system. During preperatics the Hirsch dye was used to enhance GFP glowing, which is a FRET-based effect (FRET - Förster Resonance Energy Transfer). The light wave emitted by the Hirsch dye additionally excites GFP, what happens only when the the distance between the two molecules is not greater than 10 nanometers. Infected HeLa cells can be seen as a mildly-glowing areas containing bright spots of GFP inside.
Pictures of HeLa cells transfected using our BactoDHL strain. Above are pictures in visible light, GFP fluorescence and Hirsch dye fluorescence. Below is computer generated overlay of those images. Succesfully transfected cells are significantly brighter thanks to FRET between Hirsch and GFP.
Comparision of fluorescence levels between transfected (left) and non transfected cells (right)
Flow Cytometry made it possible to express in numbers what could be seen under the microscope. For each sample during preperatics lysozyme was used to prevent the adhesion of bacteria to the surface of HeLa, but 100% efficiency cannot be expected form this procedure (HeLa Cells incubated with GFP-producing but non-invasive Top10) The results of control experiments are as follows:
Left: HeLa Cells incubated with non-invasive, non-transformed Top10 E. coli – no fluorescence observed;
Right: HeLa Cells incubated with GFP-producing but non-invasive Top10. Hardly noticeable fluorescence observed.
Left: HeLa Cells incubated with J23102+INV+LLO+GFP-transformed E. coli Top10;
Right: HeLa Cells incubated with AraC+INV+LLO+GFP-transformed E. coli Top10.
In both cases two different “populations” of GFP can be observed, depending on two varying fluorescence levels. These represent the amounts of protein (indicated by marker M2 on histograms) still entrapped in endosome and (indicated by marker M3 on histograms) distributed to cytoplasm. This interpretation is based on the results of many previous studies indicating that the GFP is able to act as a pH sensor.
The existence of relationship between pH and levels of GFP fluorescence is a commonly known fact. The Microfluidic system was used to investigate this relationship on the FACS machine used for BactoDHL-related experiments.
from: A.D. Griffiths, D.S. Tawfik Miniaturizing the laboratory in emulsion droplets, Trends in Biotechnology (2006) Vol.24 No.9
Microfluidic system, also termed in vitro compartmentalization (IVC) is a water-in-oil-in-water emulsion containing droplets as small as bacteria, having volumes of less than a femtolitre. Enclosing GFP in different pH buffers in such ‘beads’ provided the result without complicated transfection-based procedures.
The data analysis leads to a simple conclusion: The less acidic (closer to neutral) the pH, the stronger the GFP fluorescence. This strongly supports our theory: in the lyzosome (pH~5) weaker fluorescence of GFP is observed than in the cytosol (pH~7).
HeLa cells incubated with non-transformed Top10, GFP-producing Top10 and BactoDHL: percentage of GFP delivered to different compartments.
Please note the high levels of invaded cells (86,74% and 87,04% for J23102 and pAraC constructs, accordingly). In reference to control experiments (Top10), it has been proven that results up to 10% can be explained by spontaneous uptake. The result for HeLa incubated with GFP-producing, noninvasive Top 10 is explained by both spontaneous uptake and minor adhesion not eliminated by lysozyme.
Efficiency of GFP delivery to mammalian cells cytoplasm
BactoDHL proves to be highly efficient intracellular protein delivery system. GFP was detected in the cytosol of 38,96% of HeLa cells incubated with J23102 version. The number for AraC is 48,82%.