The verification of the protein content in membrane fractions after separation of Gram-negative bacterial membranes is to date a tedious and demanding process. With this project, we wanted to create Escherichia coli (E. coli) strains with a stable expression of membrane bound fluorescent biomarkers. Labeling the membranes of E. coli BL21 Gold (DE3) with fluorescent proteins allows a membrane separation to be easily verified with simple fluorescence detection. Furthermore, we wanted to investigate and compare the grade of separation obtainable with the two membrane separation techniques selective detergent treatment and density gradient centrifugation. Six fluorescent membrane labels were produced by fusing the genes encoding a fluorescent protein and a membrane protein, or an artificial signal sequence. These fusion genes were expressed from the plasmid pACYCDuet-1 in E. coli BL21 Gold (DE3), and the membranes were separated with the selective detergent treatment. This method proved to be an outer membrane (OM) enrichment technique, where the OM fraction was relatively clean, while the inner membrane (IM) fraction contained contaminants in the form of OM proteins and lipopolysaccharides. Four membrane labels were chosen for further experiments based on the transport to the intended membrane and the grade of separation obtained with the selective detergent treatment. In the next part of the project, the transport and localization of the fluorescent biomarkers to the intended membrane was verified by utilizing the density gradient centrifugation technique. This proved to require more precision and time, but gave a more complete separation of the two membranes. The biggest issue with the fluorescent biomarkers was the loss of a major part of the total fluorescence after fluorescent protein expression and separation of the membranes. We found that biologically active inclusion bodies were part of the problem, and the addition of a centrifugation step of 10 000 x g before pelleting the membranes removed these from the membrane fractions. To reduce the accumulation of inclusion bodies in the cells, a lower expression level of the fluorescent biomarkers was required. In the final stage of the project, the two most suitable fluorescent markers were to be transferred into the E. coli BL21 Gold (DE3) genome using λ red recombination. Due to time limitations, this part of the project was not completed. More experimentation and optimization is required to obtain recombinant clones with this technique.