Team:NTNU-Trondheim/Project
From 2013.igem.org
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By using multi-color fluorescence microscopy, FPs are often used in combination to examine interactions between their fusion partners. | By using multi-color fluorescence microscopy, FPs are often used in combination to examine interactions between their fusion partners. | ||
In the second part of our project, we want to create new combinations of FPs. We intend to make this happen by fusing different FPs together into a new construct, namely GFP, red fluorescent protein (RFG), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). This will give us new combinations of FPs with new absorption spectrum. The goal of this work was to expand the color palette of FPs with variants exhibiting improved brightness and contrast relative to single FP.</p><br><br> | In the second part of our project, we want to create new combinations of FPs. We intend to make this happen by fusing different FPs together into a new construct, namely GFP, red fluorescent protein (RFG), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). This will give us new combinations of FPs with new absorption spectrum. The goal of this work was to expand the color palette of FPs with variants exhibiting improved brightness and contrast relative to single FP.</p><br><br> | ||
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+ | <div class="col4" style="background-color:white;><a href="https://static.igem.org/mediawiki/2013/e/e4/FP_dimer.png"> <img src="https://static.igem.org/mediawiki/2013/e/e4/FP_dimer.png" width="400"> | ||
+ | <p style="text-align:center; color:black; "> <b>Figure:</b> Our Tat_GFP_RFP construct.</p> </div> | ||
+ | </p> | ||
+ | <div class ="row-end"> </div> | ||
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Revision as of 12:13, 4 October 2013
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 want to see if they can 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 hide
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 several properties such as quorum sensing, biofilm development, involvement in pathogenesis (some for pathogenic bacteria), DNA transfer and transport of enzymes to distal locations [1]. The OMVs contain 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:
The ability of some pathogenic bacteria to use their OMVs in pathogenesis is particularly interesting. Pseudomonas Aeruginosa and pathogenic E.coli has been studied to some extent for their OMVs role in pathogenesis. These vesicles contains virulence factors such as proteases, pro-inflammatory proteins and toxins, and attack eukaryotic cells by binding on the surface and then internalize (see figure below) [4].
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 [5]].
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: Immune cells recognize and attack vesicles.
Figure: Protein G binds Human Serum Albumin and masks the vesicles from the immune system.
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: 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[1].
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[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[3].
Figure: Fluorescent Protein Excitation and Emission Spectra.
Fluorescence is basically a color-resolved technique, to choose a right FP is important to consider its spectral profile, that is, the color of its fluorescence. A broad range of FP variants that span nearly the entire visible spectrum has been developed and optimized. For comparing the brightness of various FPs, each normalized excitation spectrum is multiplied by its peak molar extinction coefficient, and then divided by the peak molar extinction coefficient of EGFP. Likewise, each normalized emission spectrum is multiplied by its molecular brightness (molar extinction coefficient × quantum yield) and then divided by the brightness of EGFP. [1].
Choosing of the best-performing FPs in each color class are based on a number of critical factors, including maturation efficiency, spectral properties, photostability, monomeric character, brightness, fidelity in fusions and potential efficiency as a Förster resonance energy transfer (FRET) donor or acceptor.[2].
The blue, green, and yellow FPs appear to have reached their potential, but improvements are likely to continue for orange, red and combinations of different FPs. Each FP has its own defined emission and excitation spectrum.
Table: Fluorescent Protein Excitation and Emission Wavelength.
Figure: Our Tat_GFP_RFP construct.