Team:NTNU-Trondheim/Project

Gram negative bacteria export numerous proteins into the periplasm. The twin-arginine translocation pathway (Tat pathway) is a protein export, or secretion pathway found in plants, bacteria, and archaea. In contrast to the Sec pathway which transports proteins in an unfolded manner, the Tat pathway serves to actively translocate folded proteins across a lipid membrane bilayer. By using this transportationsystem we aim to introduce new proteins into the periplasm of bacteria. Once in the periplasm the protein will to some extent end up in outer membrane vesicles (OMV's) that budd of the bacteria.

As all gram negative bacteria produce outer membrane vesicles, we looked into the content of these vesicles. Was the sorting of proteins random? Could we direct certain proteins toward them? And what function would that give? Can we use this to our advantage?

Studies show that a twin-arginine signal peptide is able to direct the export of active green fluorescent protein (GFP) in E.coli and that translocation almost exclusively occur by the Tat-pathway. With this in mind we proceeded with making a construct containing tat and a GFP. As GFP's are poplular reportergenes we thought it would be fun to explore them further. As the absorbtionspectrums of different GFP's are quite similar we wondered how they might look when dimerized. As part of our project we will design a fluorescent protein dimer with GFP and red fluorescent protein (RFP). In addition we will fuse them with the tat signal peptide that will direct them into the periplasm and further to the OMV's. The FP-dimer construct will be a BioBrick. As the goal of the project is to determine if vesicles can be utilized for drug delivery we wanted to see if they could be masked from the immunesystem by introducing a specific protein.

Protein G is known to bind to human serum albumin which helps S.dysgalactiae subsp. equisimilis hiding from the immune system. Protein G could therefore be a potential important piece in a drug carrier by masking it from immunological destruction. Introducing protein G into vesicles also demonstrate that it is indeed possible to manipulate the content and therefore the properties of OMV's.

As our constructs will be put into plasmids for cloning we decided to make a new promoter BioBrick.

Gram negative (and some gram positive) bacteria produce outer membrane vesicles (OMV) by outward bulging of the outer plasma membrane. These OMV has varies function such as quorum sensing, biofilm development, involvement in pathogensis (some for pathogenic bacteria), DNA transfer and transport of enzymes to distal locations [1]. The OMVs contains different proteins on their membrane surface and within their membrane, with the most abundant proteins being OmpF, OmpC and OmpA [2]. When running a SDS-PAGE on a vesicle sample a protein distinct pattern will arise as illustrated in the figure below:

Whereas OMVs function and contents has been studied for decades, their potential as a drug carrier has not been investigated before. 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. Especially vesicles from pathogenic bacteria could be further manipulated as these vesicles naturally can attack human eucaryotic cells (see figure below)

Figure: Immunecells recognize and attack vesicles.

Figure: Protein G binds albumin and masks the vesicles from the immunesystem.

The first fluorescent protein 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 the proteins of interest. Having a variety of FPs gives scientists the opportunity to study different proteins at the same time.

The discovery of green fluorescent protein in the early 1960s eventually published 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[1].

Fluorescent proteins are in the group of structurally homologous class of proteins that they 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 has also been isolated from squeezates" (Shimomura et al. 1962). "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[2].

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.