Team:NTNU-Trondheim/Project

The vesicles will display a lot of the same proteins as the bacteria as they bulge of from the outer membrane. This trait, and the fact that the vesicles will not be able to multiply on their own, has qualified them as a possible candidate for vaccines. Engineered vesicles has been shown to be immunogenic in mice, but more research remain before OMV as vaccines can be tested on humans [4]].

Potential as a drug delivery vehicle

Whereas OMVs function and contents has been studied for decades, their potential as a drug carrier has not previously been investigated. As it is still many obstacles for an effective production method of different drug carriers within the field of nano-medicine, exploiting gram negative bacteria OMV production could be a possibility for drug delivery in the future. Making drug carriers synthetically in small sizes is difficult and the vesicles measure only 20-200 nm in size which makes them attractive for this purpose. Especially vesicles from pathogenic bacteria could be further manipulated as these vesicles naturally can attack human eukaryotic cells. The issue is that they contain PAMP's (Pathogen Associated Molecular Patterns) that are recognized by the immune system. Gram negative bacteria contain LPS (Lipopolysaccharide) in their outer membrane that is recognized as foreign by immune cells. To stop the immune cells from attacking the vesicles we propose to introduce the outer membrane protein,Protein G, into the vesicles. This protein binds Human Serum Albumin (HSA) which tricks the immune system into thinking the vesicles are harmless blood cells. This will make the vesicles more suitable as a drug delivery vehicle.

Figure 3: Immune cells recognize and attack vesicles.

Figure 4: Protein G binds Human Serum Albumin and masks the vesicles from the immune system.

Fluorescent Proteins

The first fluorescent protein (FP) to be discovered was the green fluorescent protein (GFP) isolated from Aequorea victoria. Many other different fluorescent protein (FP) with different colours (red, cyan, yellow and other version of these) has since been created by mutating GFP. The FPs has been crucial in terms of visualising cells and determining intracellular proteins by fusing the FPs to proteins of interest. Having a variety of FPs gives scientists the opportunity to study different proteins at the same time.

Figure 5: Fluorescent Proteins.

The discovery of green fluorescent protein in the early 1960s eventually created a new era in cell biology by assisting investigators to apply molecular cloning methods, fusing the fluorophore moiety to a wide variety of protein and enzyme targets, in order to monitor cellular processes in living systems using optical microscopy and related methodology[5].

Fluorescent proteins are structurally homologous proteins that share the unique property of being self-sufficient to form a visible wavelength chromophore from a sequence of 3 amino acids within their own polypeptide sequence. Biologists introduce a gene (or a gene chimera) encoding an engineered fluorescent protein into living cells and subsequently visualize the location and dynamics of the gene product using fluorescence microscopy.

The presence of a fluorescent component in the bioluminescent organs of Aequorea victoria jellyfish was introduced by Davenport and Nicol in 1955, but it was Osamu Shimomura who was the first to recognize that this fluorophore is actually a protein. He mentioned in one of his papers A protein giving solutions that look slightly greenish in sunlight though only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite that has been isolated from squeezate. Squeezate describing a solution that resulted from the squeezing of the excised bioluminescent tissues of the jellyfish through a cotton bag. After that Shimomura reported the fluorescence emission spectrum of this protein and also he suggested that energy transfer from aequorin to this green fluorescent protein could explain why the in vivo luminescence of Aequorea is greenish and not blue like the luminescence of purified aequorin[6].

The gene for green fluorescent protein was first cloned in 1992, but the important possibility to be used as a molecular probe was not achieved until several years later when fusion products were used to track gene expression in bacteria and nematodes. Green fluorescent protein has been engineered to produce numbers of various colored mutants, fusion proteins, and biosensors which are broadly referred to as fluorescent proteins. Recently, fluorescent proteins from other species are also identified and isolated, resulting in further expansion of the color palette. With the rapid evolution of fluorescent protein technology, the utility of this genetically encoded fluorophore for a wide spectrum of applications beyond the simple tracking of tagged biomolecules in living cells is now becoming fully appreciated.

A broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, and deep red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness[7].

Figure 6: Fluorescent Protein Excitation and Emission Spectra.