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Group 9 Project - Fluorescent Proteins
- 1 Introduction
- 2 The Theory of Fluorescence
- 3 The History of the Green Fluorescent Protein
- 4 Green Fluorescent Protein
- 5 Development of New Fluorescent Proteins
- 6 Current research
- 7 Advantages of Fluorescent Proteins
- 8 Limitations of Fluorescent Proteins
- 9 Links to Current Research
- 10 Glossary
- 11 References
The Green Fluorescent Protein (GFP) was first observed in the Aequorea victoria jellyfish in 1962, and since then this protein has become a vital tool for viewing many biological processes that were once invisible. The GFP can be bound on many of the thousands of different proteins in living organisms, in real time and in vivo or in vitro. Many new colours of fluorescent proteins are now available and allow multiple cell structures or processes to be imaged simultaneously. Fluorescent proteins, in their many colours and properties, have revolutionised modern cell biology as we know it, and continue to play a part in cutting-edge research into normal function and disease.
The Theory of Fluorescence
Fluorescence is the spontaneous emission of radiation (ultraviolet, visible or infrared photons) from an excited atom. The Latin word luminescenz describes all phenomena in which light is released and where a rise in temperature does not occur, whereas incandescence involves heat. The various types of luminescence are classified according to the mode of excitation, fluorescence occurs via the mode of absorption of light to release another wave of light. A prime example is the reaction of luciferase where the oxidation of a substrate by an enzyme releases light. [1] (2)
There are three principles of fluorescence
1. Excitation; energy/ photons is absorbed by the atom of an appropriate wavelength by the fluorophore which becomes excited.
2. Excited state; the electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, the atom will emit light as the atom's electron reverts to a lower, more stable, energy level, thus fluorescing.
NB; fluorescence is cyclical therefore a fluorophore usually is excited repeatedly.
Fluorescence microscopes emit a specific wavelength to excite the specimen, the microscope has a filter that only allows the specific emitted wavelength which helps make up the image without other emmited light to get through and scatter desired light. (1, 2)
The History of the Green Fluorescent Protein
Major Milestone Timeline (2, 3)
1565 N. Monardes observed the emission of light by an infusion of wood lignum Nephriticum (first reported observation of fluorescence).
1640 Licetus studied the Bolognese stone and defined it as a non-thermal light emission.
1842 E. Becquerel made the first statement that the emitted light is of longer wavelength than the incident light.
1853 G. G. Stokes introduces the term fluorescence.
1858 E. Becquerel created the first phosphoroscope.
1944 Lewis and Kasha Triplet state
1955 Green fluorescent substance in jellyfish first described.
1962 GFP identified as protein, extracted from Aequorea victoria jellyfish
1979 Osamu Shimomura characterized structure of chromophore.
1985 Prasher clones GFP gene from Aequorea victoria.
1993 The Structure of GFP chromophore confirmed, side amino acid residues corrected from Shimomura's 1979 structure.
2000 onwards The development of GFP variants, as described below in "The Development of New Fluorescent Proteins"
GFP and The Nobel Prize
The 2008 Noble Prize in Chemistry "for the discovery and development of the green fluorescent protein" was awarded jointly to:
Osamu Shimomura- When Shimomura first obtained GFP, he did so by squeezing strips of the jelly fish Aequorea through filtercel and then purified it by ammonium sulfate and column chromatography. (4)
Martin Chalfie- conducted experiments by tagging GFP in six individual cells in the transparent roundworm Caenorhabditis elegans, supported previous ideas that it can be used as a molecular tagger or "light bulb", he also contributed to the ideas of transfecting cells. With his experiment he was able to revolutionize the biosciences.
Roger Y. Tsien- -was able to mutate GFP and create a variety of new colours now termed the NEW FRUIT FP's, he was also able to make the DsRed FP compact thus more versatile, and finally he created mutants that start fluorescing faster and brighter. (5)
The Extraction and Purified of GFP from the Aequorea victoria Jelly fish (6,7,8,9)
1. Transfection into cells via vector for amplification (See Current Research section for detailed methodology)
2. Cell are lysed by sonication, then centrifuged
3. Products are put in ammonium sulfate, ethanol and chloroform and mixed it, resulting in GFP at upper aqueous phase and others proteins at bottom. Alternatively, the hydrophobic nature of the GFP molecule can be utilised for purfication with a size column
4.To confirm the extarction of GFP, the sample is excitated at 395nm (UV light) and checking if a 510nm wavelength emission results.
Further links to purification methods:[4] , [5]
Green Fluorescent Protein
Structure and Classification
GFP is compromised of 238 amino acids and weighs 26.9kDa, discovered in 1962 by Osamu Shimomura in the jellyfish Aequorea victoria, it glowed green when under ultraviolet light. Its structure was found in 1972 by Shimomura, it is an 11-stranded beta-barrel threaded by an alpha-helix running up the axis of the cylinder, it is predicated that the beta-can structure protects the chromophore and is presumably responsible for GFP's stability. This protein is now the most commonly used biological marker tool in molecular biology, medicine, and cell biology. (10, 11, 12)
The Protein Data Bank currently lists 22 GFP's, they are many mutants, this is done by changing amino acids in them, they actually all have similar structures to aequorea GFP. The chromophore is in the centre of the beta can and is a 4-(p-hydroxy-benzylidene) imidazolidin-5-one attached to the peptide backbone through the 1- and 2-positions of the ring.
Tsien (5) has classified GFPs into seven major classes based on their spectral characteristics:
(i) Wild-type GFP. The chromophore is in an equilibrium between the phenol and phenolate form. It has two excitation peaks at 395 and 475 nm.
(ii) Phenolate anion. Ser65 has been substituted with Thr, Ala, or Gly. Does not have the 395 nm excitation peak.
(iii) Neutral phenol. Mutation of Thr203 to Ile results in a mutant that only has the 399 nm excitation.
(iv) Phenolate anion with stacked ð-electron system. Mutation of Thr203 to His, Trp, Phe, or Tyr results in yellow fluorescent proteins.
(v) Indole in chromophore. Cyan fluorescent proteins have properties intermediate to those of BFP and EGFP.
(vi) Imidazole in chromophore and phenyl in chromophore. Blue fluorescent proteins have an excitation peak at 383 nm.
(vii) Phenyl in chromophore. It has the shortest excitation wavelength and no apparent uses.
|Advantages of GFP (5, 13)
|Disadvantages of GFP (5, 13)
|Applicable to nearly all organisms and in live tissue
|Gene transfection required which is time consuming
|Can be targeted to specific tissues, cells, organelles, or proteins
|GFP is most efficient temperatures well below 37 °C, thus searches for ones functional in mammalian cells were made.
|Unlikely to diffuse well enough to blur spatial gradients
|GFP only fluoresces in presence of oxygen as the chromophore requires an autocatalytic reaction, thus GFP only works in aerobic environments.
|Good optical properties: visible excitation, high photo-stability
|Some of GFP's limitations are the slow post-translational chromophore formation.
|Cheap and relatively easy to replicate and distribute
|Difficulty in distinguishing GFP from background fluorescence when the GFP is not densely localized or highly expressed.
|It's chromophore does not require a cofactor
|It is highly stable as GFP is resistant to heat, alkaline pH, detergents, photo-bleaching, organic/ non-organic salts, and many proteases.
|Shimomura showed GFP was a relatively small protein which allows little hindrance to the protein or its function.
Tagging GFP onto Targets
GFP is so versatile and can be tagged by means of labelling reagents with functional groups on amino groups via covalent binding, Wang and Hazelrigg discovered that GFP can be used as a fluorescent tag for the N- or C-termini of proteins. Since there are so many proteins it can labelled on nearly all aspects of the cell. (5,12,14)
Development of New Fluorescent Proteins
Since the revolutionary development of GFP, extensive research has been conducted with the aim of producing fluorescent proteins (FPs) which cover a broader colour spectrum. These colour mutant FPs are also being genetically engineered to exhibit faster maturation rates, greater photostability, increased brightness, pH insensitivity and reduced oligomerisation and toxicity (15)(16).Due to the sheer number of FPs that have been produced, this section will focus on the most current and valuable colour mutant FPs and touch on FPs with other specialisations such as photoactivation and photoconversion.
New FP Colours
Enhanced BFP (EBFP) was one of the first spectral variants engineered from Aquorea GFP, but due to its low brightness and poor photostability it is now unappealing for most research (15) (17) (18). EBFP2 is the most photostable and brightest blue FP (15).
The cyan FPs (CFPs) began with the production of enhanced CFP (ECFP) from Aequorea GFP (19). mCerulean followed in 2004 offering a brighter and better general-purpose CFP (20). In 2006, a monomeric teal-coloured variant mTFP1 was obtained from a Clavularia soft coral protein (21). mTFP1 is brighter, less pH sensitive and more photostable than the traditional CFPs, making it an excellent alternative to its predecessors (15).
Most recently, site-directed mutagenesis of ECFP has produced super CFP (SCFP) which is twice as bright as ECFP when expressed in bacteria. SCFP shows promise for use as a fusion tag or as a biosensor for the detection of calcium ion fluctuations, pH changes, metabolites or enzyme phosphorylation (15). Other proteins which fall into this class include mCFPm (22) and CyPet (23).
Following the discovery of the original Aequorea GFP discussed previously, many other proteins which express in the green region of the spectrum have been isolated from other Aequorea species, copepods, amphioxus and reef corals (15).
Most of these novel GFPs exhibit no discernable advantage over EGFP (15) and hence will not be discussed. mEmerald, a derivative of EGFP, is currently the best choice for live-cell imaging due to its more efficient folding than EGFP at 37°C (15)(24).
Yellow FPs (YFPs) are among the brightest and most versatile probes developed in any of the spectral classes (15). EYFP was developed in 1999 (26) and is still widely used despite its high pKa and sensitivity to halides (15). EYFP has proved effective in tracking the distribution patterns of single proteins on the membranes of live cells (27). mCitrine, derived from the addition of a single mutation in EYFP, is less halide sensitive and twice as resistant to photobleaching as its predecessor [2].
mVenus is a popular YFP mutant with a greatly reduced maturation time, however it has low photostability. The fact that mVenus requires only two minutes in vitro or seven minutes in vivo to produce fluorophores makes it ideal for monitoring cellular processes with fast dynamics such as gene expression [3].
Super YFP (SYFP), the product of site-directed mutagenesis of EYFP, may hold similar applications as those discussed previously for SCFP. Yellow fluorescent protein for energy transfer, YPet, is the brightest YFP variant. YPet is also reported to have very good photostability and superior acidic resistance to mVenus and other YFP derivatives [4].
Compared to the other areas of the spectrum, few probes have been constructed to emit in the orange and red wavelengths. Probes such as DsRed, TagRFP and tdTomato actually have emission profiles in the orange range of the spectrum and not the red range as suggested by their names (15). mOrange, a member of the 'Fruits' series (see 'Red' section for details), once dominated this spectrum in terms of brightness, but has average photostability and is unstable at low pH (15). In an attempt to overcome these problems mOrange2 was engineered from mOrange and shows significantly improved photostability but is still pH sensitive and shows an almost doubled maturation time.
Kusabira Orange (KO), derived as a tetramer from the mushroom coral Fungia concinna, was later modified to give monomer KO (mKO) (30). mKO demonstrates extremely good photostability and brightness similar to that of EGFP, making it a good candidate for long-term and wide-fluorescence illumination experiments. (15,31)
In 2007 TagRFP was cloned as a dimer from Entacemaea quadricolor sea anemone and appears to be a useful tool for localisation and FRET studies (32). Random mutagenesis of TagRFP produced the highly photostable, bright and pH resistant TurboRFP.
Many of the problems associated with Discosoma DsRed FP- including slow maturation, an intermediate green state, and tetrameric character- have been the target of many attempted modifications of this protein through both random and site-directed mutagenesis. The production of the monomeric mRFP1 from DsRed was promising, but reduced emission and quick photobleaching still means that it is less useful than monomeric GFPs and YFPs. (33)
tdTomato, another of the 'Fruits' proteins, is the brightest of all available FPs, emits at closer to the true red range and is very photostable (31). The major drawback in the use of tdTomato is its comparatively large size which is proposed to interfere with fusion-protein packing (15).
FPs that emit in the far red area of the spectrum are desirable due to this wavelength of light being less phototoxic and more able to probe deeper into biological tissues (15). The most promising develops have arisen from the site directed mutagenesis of mRFP1 to give monomeric FPs which emit in the 560-610nm wavelength which are collectively referred to as the 'Fruit' proteins (15). Despite their improved emission colours, many Fruits lack the brightness and photostability needed for most experiments (16). According to Shaner and team, mStrawberry and mCherry are the best reds, with brightness levels of 75% and 50% of EGFP (15). mCherry is more photostable than mStrawberry (16) and as well as a better alternative to mRFP1 for long-term imaging experiments (15).
Developed in 2004, mPlum is one of the first true far-red probes, emitting at 649nm (34). mPlum has limited brightness (10% of EGFP) but good stability and is recommended for use in multicolour imaging experiments, the imaging of thicker tissues and as a FRET partner for GFPs and YFPs (31)(15).
Kutushka, a dimeric protein that emits at 635nm, was developed in 2007 and is commercially available from Evrogen as TurboFP635 (35). Despite being less bright than EGFP, Kutushka has the highest brightness level of any of the FPs in the 650-800nm wavelength area.
mKate (Evrogen, TagFP635) has similar spectral characteristics to Kutushka, brightness on par with mCherry and is reported to be very photostable, making it a good candidate for localisation experiments in this area of the spectrum (35). mKate has been reported to exhibit complex photobleaching behaviour which is yet to be well characterised and it is suggested that mCherry remains a more reliable choice for single-molecule imaging (15).
For more information on the range of currently available colour palette of FPs visit:
- The Clontech "Interactive Guide" (found as a link on the righthand side of the page)- allows you to explore their range of comercially available FPs by colour, application, spectra and antibody choice [6]
- The Nikon Microscopy Education page- gives the basics of FP use in live cell imaging [7]
- The Olympus FluoView Resource Centre- gives information on the colour palette for confocal microscopy [8]
Other Novel FPs
Infra-Red FPs
The development of a protein that emits in the infra-red spectrum is the holy grail of fluorescent protein engineering. Accurate in vivo imaging of animal tissue using FPs requires emission wavelengths of 650 to 900nm to minimise absorbance by water, lipids and haemoglobin and to reduce light scattering. Infra-red fluorescent proteins (IFPs) which can be genetically expressed would be incredibly valuable for whole-body imaging in cancer and gene therapy (15).
A paper published in 2009 (36) reports the production of a monomeric IFP from a bacteriophytochrome which incorporates biliverdin (an intermediate of heme catabolism) as the chromophore. The IFP was effectively expressed in both mammalian cells and mice, demonstrated the spontaneous incorporation biliverdin and produced infra-red fluorescence.
Photoactivated FPs
These proteins display negligible fluorescence until excited by irradiation at a specific wavelength (37). This allows for the highlighting of molecules within a discrete region of a cell as well as a way to study the lifespan and behaviour of proteins independently of other newly synthesised proteins (37). PA-GFP is one such protein that is activated by light of wavelength 488nm and emits in the green wavelength (38). Other FPs which demonstrate "reversible on/off switching" are Dropna, rsFastLime, rsCherry, rsCherryRev and PAmCherry1 (38).
Photoconvertable FPs
This class of FPs show "light-driven modulation of fluorescence properties" (38) and demonstrate green-to-red conversion when activated at around 400nm (38). These proteins, such as Kaede, KikGR, EosFP, Dendra2, mKikGR, tdEosFP, mEosFP and mEosFP2, are useful for the tracking of fusion proteins, organelles or the fate of embryonic cells during development (15,38).
Destabilised GFP variants
These FPs allow the characterisation of the expression timing or lifetime of a target protein due to their rapid turnover by proteolysis resulting in only younger protein chimeras fluorescing (39).
"Fluorescent Timer" Protein
This protein, similarly to the destabilised GFP variants, allows the measurement of protein turnover and expression timing (37). It initially fluoresces in the green area of the spectrum before conversion of the fluorophore after several hours leads to emission in the red (40). Using the ratio of green to red fluorescence allows the age of the tagged protein to be determined.
Future Engineering Endeavours
Criteria for future engineering endeavours as proposed by Shaner et al. (16):
• Improved brightness
• Monomeric structure
• High contrast
• Uncomplicated photoconversion
• Reversible photoactivation
• Red-to-green photoconversion
• Improved expression in the far-red or near-infrared regions
Current research
Current research in cell biology with fluorescent proteins is a vast field of study. We shall focus our investigation on a specific technique that has significant clinical consequences. This technique is the production of individual transgenic cells and whole transgenic organisms using spliced fluorescent genes. The principle behind this technique is based on the model for protein synthesis and thus an explanation of the protein synthesis model is an essential requisite. Protein synthesis occurs in 3 stages:
The first stage: (Transcription)
1) Inside the nucleus of a cell, the double stranded DNA helix unwinds in the area of the gene containing the information for synthesizing a specific protein (41,42).
2) The enzyme RNA polymerase moves along the unwound DNA strand at the location of the gene using it as a template to create a complementary strand known as a messenger RNA strand (mRNA). This complementary strand is made by linking RNA nucleotides that are complementary to the gene nucleotides on the DNA strand. Note: In any RNA strand, the base thymine is replaced with uracil and the start codon (AUG) and a stop codon (UAA or UAG or UGA) control the length of the strand (41,44).
3) The mRNA strand then undergoes modifications in the nucleus to contain only the base sequence that will code for the protein. (42) Most genes contain non-coding regions known as introns and coding regions called exons (41,42). Thus its complementary mRNA strand will also contain coding and non-coding regions. The non-coding regions are excised by splicing introns from the mRNA and joining the exons together (41,42).
The second stage: (Amino acid activation)
This stage occurs in the cell cytoplasm where the aminoacyl-tRNA synthetise enzyme attaches amino acids to transfer RNA molecules (tRNA). Each type of amino acid is attached to its specific tRNA (44).
The final stage: (Translation)
1) The mRNA strand binds onto a ribosome in the cytoplasm at the end with the start codon (AUG) (41,44).
2) A tRNA molecule binds to the start codon on the mRNA within the ribosome (44).
3) A second tRNA binds to the next codon of the mRNA within the ribosome (44).
4) The first tRNA is released from the ribosome and the ribosome moves along the mRNA strand one codon at a time (42). Two tRNAs are temporarily bound within a ribosome at any one time and their amino acids linked together forming a polypeptide chain. This process ends when the stop codon is reached on the tRNA molecule and the polypeptide is then released (42,44).
5) The polypeptide then undergoes further modifications in the cell cytoplasm particularly in the Golgi apparatus before becoming the final protein (42).
Transgenic Organism
A transgenic organism is one whose genome contains genes from another species (42). The aim of creating a transgenic organism is to obtain a favorable characteristic in the organism's phenotype. This desired characteristic of phenotype is obtained by altering an organism's normal genotype to include the gene from another species coding for the desired characteristic (44). Once a desired gene is incorporated into an animal's genotype, the process of protein synthesis allows the transgenic organism to express this new characteristic in its phenotype (42,44).
In the case of fluorescent research, the gene responsible for fluorescence phenotypic characteristics in one organism can be spliced and inserted into the genotype of another organism for experimentation. The recipient organism is then able to produce the fluorescent proteins (coded for by the inserted gene) in its phenotype (43).
The production of a fluorescent transgenic organism or cell involves several steps summarized below:
1) Organisms containing fluorescent proteins are identified. The chromosome and the gene coding for the fluorescence are identified in the organism (42).
2) A cell sample is taken from the organism and the gene coding for fluorescent protein synthesis is isolated from its DNA strand. This involves "cutting" the gene out of its DNA strand using enzymes called restriction endonucleases. The restriction endonucleases cut DNA at specific site so the desired gene can be removed from the DNA strand. The cut ends are known as "sticky ends"(41,42).
3) The gene is then incorporated into a recipient organism's DNA. To do this, the recipient organism's cell DNA must first be cut with the same restriction endonucleases at a desired location (usually just before the stop codon at the end of a gene coding for a particular protein) (41). The foreign gene is then inserted with a promoter sequence by microinjection or vector transfer into the fertilized egg cell (zygote) and as the embryo develops, the entire organism will become transgenic (43). Alternatively, the foreign gene can be injected into a somatic cell or sex cell of a post natal recipient organism, this in effect will result in the fluorescence of a particular type of cell and thus tissues rather than the entire organism (41,43).
4) The inserted foreign gene will be attracted to and connect at the recipient's cut DNA in a process called "annealing"(44). The DNA is now known as recombinant DNA as it contains a combination of the recipient's normal genes as well as foreign genes (42).
5) DNA ligases are added to the annealed DNA fragments to help strengthen the bonds of the new recombinant DNA (42,44). DNA ligases are sealing enzymes found in all living organisms that help make and repair DNA (42).
6) As the cell develops and its genetic information read, the recombinant DNA will not only code for the production of the normal protein, but will also code for the production of the fluorescent protein that will be attached to the normal protein (42). This will be true of the recipient cell and all its descendants (42).
The creation of transgenic organisms with the capability to produce fluorescent proteins has significant clinical consequences particularly in the understanding of inexorable diseases such as cancer and malaria.
Cancer is the leading cause of death worldwide claiming approximately 8 million people in 2009. The WHO estimates this figure to rise to 12 million by 2030 (46). In Australia, cancer claims approximately 42,000 lives every year and an estimated 114,000 new cases are expected in 2010 (45). A cancer or malignant neoplasm is a class of disease in which cells display uncontrolled mitotic divisions, invasion of surrounding tissues and metastasis (spread via the circulatory or lymphatic system) (47). Cancer cells are unspecialized and have no other function other than reproduction. These cells invade tissues and organs diminishing their functioning gradually until failure (43).
Fluorescent proteins provide a means by which cancer can be studied in vivo. The implications of this are significant as it allows researchers to study the characteristics of this disease as it develops in the body over time (48). A histologically intact primary tumour from a cancer patient is obtained by surgical means and transfected with a specific fluorescent protein. The transfected tumour cells are then transplanted into immunodeficient (nude) transgenic mammals (usually mice) which themselves have been genetically modified to display a different coloured fluorescent protein (48,49). This creates a contrast between the healthy cells of the recipient organism and those of the tumour cells as both display different coloured fluorescence (50,51). The fluorescent protein fluoresces for the entire life of a cancer cell and after it divides mitotically, fluoresces in the daughter cells (50). Using this contrast, researchers are able to visualize, in real time, important aspects of cancer in living animals, including tumour cell angiogenesis, mobility, invasion and metastasis (49,51).
This technique of studying cancer has so far yielded some interesting results. GFP expressing tumour cells reveal that angiogenesis occurs early in tumour formation when as little as 60 to 80 tumour cells are present inducing vasodialation and changes to blood vessel morphology (52,53). Armed with this knowledge, researchers can experiment with various methods to stop the ability of tumour cells to recruit blood vessels that are vital for their growth and spread thereby stopping the cancer in its tracks (54). Research by Yamauchi and Yamamoto explored the mechanism by which tumour cells migrate down through narrow capillaries. By expressing red fluorescent proteins in the tumour cell cytoplasm and green fluorescent proteins in the cell nucleus they showed that tumour cells are highly motile and capable of changing their morphology to great extents in order to squeeze down narrow vessels (55,56). This is critical information on the metastatic ability of cancer cells and explains to some degree the mechanism by which cancer cells are able to spread to body extremities (55).
Malaria is the world's most serious infectious disease and is responsible for one million deaths annually worldwide (57). There are currently between 250 to 500 million people affected by malaria most of them in developing countries (42,57). In Africa, malaria is an especially serious problem where a child dies from it every 30 seconds (57). In Australia, malaria was declared eradicated in 1981 but approximately 700 to 800 cases occur each year from travellers infected elsewhere with northern Australia the primary receptive zone for malaria transmission (58). Malaria is a parasitic infection caused by four species of the genus Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. The infection begins when Plasmodium sporozoites enter dermis from the bite of an infected female Anopheles mosquito (41,42,44,58). The parasites then invade blood vessel and travels by blood to the liver where they enter liver cells (hepatocytes) (41). Inside a hepatocyte, the Plasmodium sporozoites divide asexually forming Plasmodium merozoites (62). The merozoites are then released into the circulatory system where their invade red blood cells (erythrocytes) (62,63). Once inside erythrocytes, the merozoites continues to proliferate causing each infected red blood cell to burst releasing toxins into the blood stream (63). Some erythrocytes do not burst as they contain merozoites that do not proliferate but instead develop into male and female gametocytes. When a female Anopheles mosquito bites an infected host, these gamatocytes enter the mosquito abdomen where they form a zygote and develop into new active Plasmodium sporozoites that can begin a new infection cycle (63). Growing Plasmodium resistance to anti-malarial medicines such as chloroquine and vector resistance to mosquito pesticides has spread very rapidly undermining malaria control efforts across the globe (57).
Fluorescent proteins provide a unique method of imaging the entire malaria parasite lifecycle from development to transmigration inside both insect and vertebrate hosts. This is achieved by transfecting the malarial parasites with genes encoding for green-fluorescent protein (GFP) or red-fluorescent protein (RedStar) creating a transgenic parasite (59). Fluorescent microscopy can then be used to viasulize the parasites behaviour inside the mosquito vector and also in cells of the vertebrate host. Using this technique, Rogeiro et. al found that Sporozoites are formed inside oocysts at the mosquito midgut wall. They are then released into the hemolymph and eventually bind to the mosquito's salivary glands, which they then invade. When the mosquito bites its host, the sporozoites are released into the hosts' circulatory system (61). Tracking the movement of the malarial sporozoites inside the mosquito vector helps researchers understand the behaviour of the parasite and devise methods of preventing transmission to humans by stopping the parasite's advance at the vector level (59,61).
Another research focusing on control of malaria found that transgenic mosquitoes were less susceptible to plasmodium infection and had fewer sporozoites in their salivary glands than control mosquitoes. They also found transgenic mosquito parasite transmission was reduced by more than twofold. The team investigated this result to be a consequence of the expression of the SM1 peptide in the mosquito midgut that severely reduced vector competence by inhibiting Plasmodium development (60). This suggests that gene modifications can inhibit malarial sporozoite development in the mosquito and thus holds great potential for gene experimentation methods to control malaria. Other research has focused on breeding experiments where fluorescent proteins are used to differentiate male and female mosquitoes. Since female mosquitoes only mate once in their lifecycle and are the transmitters of the malaria parasite, sterile males (marked by fluorescent gonads) are sorted and released into the environment where they mate with the infected females. Since the female will be unable to reproduce, the malaria parasite life cycle is ended (64).
Advantages of Fluorescent Proteins
Some advantages in using fluorescent proteins include:
• The imaging of structures, cells, tissues, organs and even living organisms in vivo.
• There is no known adverse disruption to the normal functioning of the cell biochemistry.
• Fluorescent proteins are easily obtained from fluorescent organisms mainly corals, jellyfish and marine invertibrates.
• The isolation and transfection of fluorescent proteins is a higly successful process that produces minimal hazardous wastes unlike other fluorescent techniques like radioactive dyes and fluorescent nano dots.
• FP's are available in a vast array of colours covering different emission spectra for imaging different tissues.
Limitations of Fluorescent Proteins
Problems associated with FPs (16,38):
• Oligomerisation
• Aggregation
• Cytotoxicity due to FP
• Environmental sensitivity
• Light-induced cytotoxicity
• Potential biological activity exerted by FPs
• No strict criteria under which the FPs are tested- results are often biased and therefore not trustworthy for researchers
• Commercial availability/price
Links to Current Research
AntiCancer Inc. ((www.anticancer.com))
Bio medicine (www.bio-medicine.org)
Brighter Ideas Inc (www.brighterideasinc.com)
Central caribbean marine institute (www.reefresearch.org)
Clontech (www.clontech.com)
Evrogen (www.evrogen.com)
GeneBrite (www.genebright.com)
Glen Research (www.glenresearch.com)
MetaMouse (www.metamouse.com)
Invitrogen (www.invitrogen.com)
Promega Corporation (www.promega.com)
Angiogenesis: The formation of new blood vessels
Chromophores: Molecules which are able to absorb light
Codon: A specific sequence of three adjacent nucleotides on a strand of DNA or RNA that specifies the genetic code information for synthesizing a particular amino acid
Erythrocytes: Red blood cells
Exons: A segment of a gene that contains a coding region. The exons provide the instructions for making a protein
Filtercel:An industrial filter used to seperate small particles from big ones.
Fluorochromes or fluorophores: Molecules which are able to absorb and emit light Filtercel
FRET: Fluorescence Resonance Energy Transfer
Gametocyte: A eukaryotic germ cell that divides by mitosis into other gametocytes or by meiosis into gametids during gametogenesis
Genotype: The genetic makeup of an individual
Golgi apparatus: An organelle in the cytoplasm that packages proteins and carbohydrates into vesicles for export from the cell
Hemolymph: A circulating fluid in the bodies of some invertebrates that is the equivalent of blood
Introns: An intron is a DNA region within a gene that is not translated into protein
Ligase: Any of a group of enzymes that catalyze the binding of two molecules
Merozoite: Cell that arises from the asexual division of a parent sporozoan during its life cycle
Metastasis: When cancer cells have broken away from the primary tumor and spread to other organs in the body through the blood stream or the lymphatic system
mRNA: The RNA transcript of a protein-encoding gene
Phenotype: Phenotype is any observable characteristic or trait of an organism
Photoactivation:The absorption of energy from a photon in raising a chromophore from the ground state
Photoconversion: The photochemical conversion of a chromophore, resulting in a change in emission spectrum
Photostability: The stability of a chromophore during excitation
Random mutagenesis: Random incorporation of mutations in DNA
Restriction Endonucleases: An enzyme that cuts double-stranded or single stranded DNA at specific recognition
Ribosome: Ribosomes are the components of cells that make proteins from amino acids
Site-directed mutagenesis: Targeted insertion of a mutation in a DNA sequence, usually by use of of a plasmid vector
Sporozoite: Slender, spindle-shaped organism that is the infective stage of malaria
tRNA: A small RNA molecule that "translates" a codon in mRNA by bringing in its corresponding amino acid during protein synthesis
Zygote: A fertilized egg cell, it is diploid
- ↑ UCLA fluoresence
- ↑ Griesbeck, O, Baird, G S, Campbell, R E, Zacharias, D A & Tsien, R Y. (2001). Reducing the environmental sensitivity of yellow fluorescent protein: Mechanism and applications. Journal of Biological Chemistry, 276, [1]
- ↑ Nagai, T, Ibata, K, Park, E S, Kubota, M, Mikoshiba, K & Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell biological applications. Nature Biotechnology, 20, [2]
- ↑ Nguyen, A W & Daugherty, P S. (2005). Evolutionary optimization of fluorescent proteins for intracellular FRET. Nature Biotechnology, 23, [3]
- http://www.ucla.edu/, http://vohweb.chem.ucla.edu/voh/classes%5CSpring08%5C156ID35%5Cfluoresence.pdf.
- Book for history
- http://www.conncoll.edu/ccacad/zimmer/GFP-ww/timeline.html
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- Jennifer Lippincott-Schwartz,* George H. Patterson (2003). Development and Use of Fluorescent Protein Markers in Living Cells Science 4 April 2003: Vol. 300. no. 5616, pp. 87 – 91. http://www.sciencemag.org/cgi/content/short/300/5616/87
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2010 Projects
Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer


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Sporting KC Trades Midfielder Thomas to Toronto FC
KANSAS CITY, Kan. (AP) - Sporting Kansas City traded midfielder Michael Thomas to Toronto FC on Tuesday for a second-round pick in the 2015 MLS SuperDraft.
The 25-year-old Thomas signed with Sporting KC in January 2012 and played in seven MLS matches for the club. He also made two starts in U.S. Open Cup competitions, including a 3-0 victory in the quarterfinals of last year's championship run.
Thomas grew up in nearby Olathe, Kan., and was a star at St. Thomas Aquinas High School before playing his college soccer at Notre Dame. He began his pro career in Sweden.
Sporting KC manager Peter Vermes said in a statement that "this decision was of mutual consent. Toronto is a team that was very interested in him and we wish him all the best."

subset: commoncrawl


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Getting Around
Spend a day in San Diego, and it quickly becomes apparent why it's known as "America's Finest City." Learn more about campus and San Diego, as well as the services we offer to help you get here.
On Campus
Campus Maps
Use the campus map to find popular locations on campus. The map also includes a clickable Google map with tram stops. Our campus skateboarding map also has information about bicycle and skateboard paths around campus.
USD's Tram Service is free to all students, employees and visitors to the university and makes regular stops throughout campus. The tram also provides transportation to the Old Town Transit Center, which provides convenient access to much of the greater San Diego area.
Check out our Tram locator to find out where your tram is.
Parking Permits
Visitors are required to display a valid permit Monday through Friday, between the hours of 7 a.m. - 9 p.m. Visitor permits may be obtained from the west entrance kiosk. Visitors are allowed to park in the West Parking Structure and Lot, and the West Gravel Lot. Please refrain from parking on the main campus. Paid parking may be available in the Mission Parking Structure. For more information parking on campus, please visit the Parking Services website.
Off Campus
ZimRide is one of the many carpooling options USD sponsors to promote a more sustainable lifestyle and to reduce our carbon footprint.
Zip Cars
There are also four Zip Cars on campus available to be rented by the hour.
Public Safety and Parking Services are also available to give rides to students, if the need arises. You can contact the USD Public Safety dispatcher at (619) 260-7777 to request a ride at anytime.
Getting to USD
By Car
From the North: Use I-5 south, exit at Sea World Drive and Tecolote Road and proceed left at the stoplight onto Tecolote Road. Turn right on Morena, left on Napa and left on Linda Vista Road. Travel east on Linda Vista Road up the hill to USD's main entrance and turn left into campus.
From the South (Or the Airport): Use I-5 north, exit Morena Blvd. (signs will say "Morena Blvd. use I-8 east"). Stay to the right and follow the signs for Morena. When the road splits, stay to the right onto Linda Vista Road. Travel east on Linda Vista Road up the hill to USD's main entrance and turn left into campus.
From the East: Use I-8 west, exit at Morena Blvd. When the road splits, stay to the right onto Linda Vista Road. Travel east on Linda Vista Road up the hill to USD's main entrance and turn left into campus.
By Plane
San Diego International Airport at Lindbergh Field is located downtown and is about 15 minutes from the USD campus. Airport shuttles, taxis and car rentals can be found at all terminals.
For more alternative transportation options, visit our Parking Services website.
By Public Transportation
San Diego Metropolitan Transit System (MTS) buses, trolleys and coasters provide convenient transportation throughout San Diego County. If you want to use the local bus service, use the MTS Google Transit Trip Planner to map routes directly from USD. We are located on the #44 bus route. Bus schedules differ, but service the stop by our entrance every 15 to 20 minutes. MTS Student Passes can be purchased right here on campus at the Hahn University Center Ticket Office for a discount. iCommuteSD is another great resource to plan trips around San Diego using public transportation.
The San Diego Trolley runs every 15 minutes throughout most of the day and every 30 minutes in the evening. The stop nearest our campus is the Old Town Transit Center on the Blue/Green Line. The USD Tram runs every half-hour from Main Campus to the Old Town Transit Center.
The North County Coaster runs 43 miles along the California coastline with station stops in downtown San Diego, Old Town, Solana Beach, Encinitas, Carlsbad and Oceanside. It is best to transfer from the Coaster to a bus at the Old Town Transit Center to reach our campus.


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Cato Challenges the Supreme Court to Decide that Congress Doesn't Have Unlimited Jurisdiction Over Everyone
Last year's partial victory in the Obamacare case is already being applied to new cases reaching the Supreme Court. Recall that, in that case, the Court accepted our argument that the government cannot use the Commerce and Necessary and Proper Clauses to compel someone to purchase health insurance. The Court held that allowing Congress to compel commerce into existence would be an improper use of a great and limitless power. In United States v. Kebodeaux, the Supreme Court will once again address an assertion of power that, if upheld, could give Congress nearly limitless power.
In 1999, Anthony Kebodeaux was sentenced to three years in prison for statutory rape. He served his time, was freed from any post‐release parole or probation requirements, and ended his relationship with the federal government in the matter of criminal law. Years later, when Kebodeaux moved intrastate from San Antonio, Texas to El Paso, Texas, he failed to update his change of address within the three‐day period as required by the federal Sex Offender Registration and Notification Act (SORNA) of 2006. Even though Kebodeaux was unconditionally released from custody before SORNA was enacted, he was sentenced to one year in federal prison. The Fifth Circuit overturned his conviction en banc, meaning that every judge on the Fifth Circuit heard the case rather than the traditional three‐judge panel. They found the registration requirement unconstitutional because Congress lacked jurisdiction over Kebodeaux after they unconditionally released him from custody.
The government's arguments to the contrary, the court held, would permit not just "unending criminal authority" over Kebodeaux but unending authority over every American who was once in federal jurisdiction, which is, of course, every American.
In a sense, the government is now arguing for the "Hotel California" theory of jurisdiction: you can check out, but you can never leave.
Yesterday, Cato filed an amicus brief, joined by Ilya Somin, Professor of Law at George Mason University School of Law, arguing that it would be improper under the Necessary and Proper Clause to permit Congress to have unending authority over all Americans. Congress already lacks a general power to punish criminals, much less monitor previously released criminals and impose new and onerous restrictions on them at will. Moreover, there is nothing constitutionally special about sex offenders as a class. Congress should not be allowed to designate a sub‐class of people within its jurisdiction as "special" and then assert perpetual jurisdiction over them. These type of assertions of power are precisely what the "proper" element of the Necessary and Proper Clause is supposed to protect against–ones that, even if "necessary," would give Congress unbounded power.
Indeed, if the Court rules in favor of the government's position, it will give Congress virtually unlimited power to regulate nearly all Americans. In essence, it would justify the gradual imposition of endless new requirements on anyone who had previously been subject to federal jurisdiction. Cumulatively, these federal impositions amount to unlimited federal authority over anyone who has ever been held in federal custody or otherwise in federal jurisdiction. This cannot be a power vested in a Congress with "few and defined" powers. As the Supreme Court held in the Obamacare case, Congress doesn't have the power to "regulate an individual from cradle to grave."


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Industry safety (B.C.'s Restart Plan, Phase 1): Agriculture, Community social services (residential and non-residential), Construction, Forestry, Health care, Hospitality, Manufacturing (see also: Meat processing), Municipalities, Retail, Small business, Transit, Transportation
Returning to safe operation (B.C.'s Restart Plan, Phases 2 & 3): General guide to reducing risk; Accommodation; Arts and culture; Child care and day camps; Education (K-12); Education (post-secondary); Faith-based organizations; Gyms and fitness centres; Health professions; In-person counselling; Motion picture and television production; Offices; Parks, camping, and tourism; Performing arts; Personal services; Real estate; Restaurants, cafes, pubs, and nightclubs; Retail; Sports and recreation; FAQs; What workers should expect
Resources: COVID-19 Safety Plan template, OHS guideline G3.3 (COVID-19 Safety Plan), Ventilation FAQs, and more resources
Additional information: General health and safety; Premiums waived for Canada Emergency Wage Subsidy, Q1 and Q2 reporting and payment deadlines; Claims
2021 preliminary rates
Learn about the 2021 preliminary rates; Register for virtual rate consultation sessions; Find your classification unit, industry or rate
Forklift Safety: Stay in the Cab, Stay Alive
Toy Version
Every year workers are crushed, maimed, and killed when they jump or are thrown from forklifts. This video reminds forklift drivers to wear their seat belts. If your machine does tip brace yourself and stay in the cab.


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Free calls from Mobile & Landline numbers.
Insight Investigations get results for investigations in Southampton and the surrounding areas. Whether you are worried about a partner or your company's assets or staff, our multi-talented team have expertise in private, corporate, commercial and legal investigations in Southampton, Hampshire and across the South East. We provide a 24 hour service 7 days a week to respond swiftly to your private investigation needs.
Insight Investigations are one of the leading private investigator companies in the UK and over the last 30 years we have handled a vast range of private investigation cases in the Southampton area including:
- Matrimonial and relationship investigations if you are worried that a spouse or partner maybe having an affair.
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Insight Investigations has a strong presence in the Southampton area and has established an excellent reputation for getting results and providing clients with the peace of mind that their case will be dealt with in a discreet, sensitive and highly confidential manner, by an assigned case manager.
We have a team of highly experienced multi-disciplined male and female private investigators who are well trained in all aspects of private investigation. Their dedication to achieving results and getting a resolution to your case is their main priority and they will work closely with you to ensure that you are obtaining the best results for your investigation.
Our team of Southampton private investigators cover the whole of the city and all surrounding areas of the county of Hampshire including Bournemouth, Salisbury, Winchester, Swindon, Reading, Portsmouth, Havant, Aldershot, Andover, Basingstoke, Eastleigh, Fareham, Farnborough, Gosport, Lymington, Petersfield, Romsey, Ringwood, Southsea, Tadley, Waterlooville, Wickham and Yateley.
With over 30 years of experience and great local knowledge of Southampton, Insight can offer unrivalled investigation services in Southampton for private, corporate, commercial and legal clients alike. Our centrally located office is the ideal location from which to operate and ensure we can carry out your private investigation swiftly, discreetly and provide excellent results.
Call our Southampton office on
0800 14 55 123
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Insight Investigations
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MINNEAPOLIS, May 14 2014—The Walker Art Center is thrilled to present Art Expanded, 1958–1978, drawn primarily from the Walker's diverse collection of artworks, films, archival materials, and ephemera, on view from June 14, 2014 through March 8, 2015. Charting the so-called "expanded arts" of the 1960s and 1970s, the exhibition surveys a transformational phase in the history of 20th century art when artists around the world collectively began to challenge, critique, and upend traditional media and disciplines. This catalytic shift in attitude and practice, which remains vital for contemporary artists working today, led to a proliferation of new, hybrid art forms, such as happenings, event scores, intermedia performance, multiples, conceptual art, and participatory environments. Related events include the overnight opening on June 14 for Northern Spark, a re-staging of Alison Knowles' event score Make a Salad on July 10, and a gallery talk with exhibtion curator Eric Crosby on July 31.
Some 300 works by over 100 pivotal artists, filmmakers, and choreographers will be presented, including Yoko Ono, Jasper Johns, John Cage, Allan Kaprow, Nam June Paik, Trisha Brown, Merce Cunningham, George Brecht, Yayoi Kusama, Yvonne Rainer, Adrian Piper, Dieter Roth, and Tony Conrad. Iconic pieces by these and many others artists in the Walker's collection will come together with recent acquisitions, rarely seen works, and newly conserved sculptures. From room-based installations of objects and archival ephemera to an array of projected images and sound works, the exhibition will be organized into thematically groupings that occupy over 13,000 square feet in Galleries 1, 2, 3 and Perlman.
"No longer content to work within the parameters of conventional disciplines, artists of the period advanced radical ways of working in the spaces between traditional mediums, many of them ultimately embracing the creative potential of everyday life," explained Walker curator Eric Crosby. "Art Expanded, 1958–1978 not only examines this critical point of departure, but also narrates a history of our present moment—so much of the work on view here has become the very DNA of contemporary artistic practice."
Among the overlapping themes of the exhibition, Art Expanded, 1958–1978 will consider the period's many assaults on the traditional mediums of painting and sculpture. Exploring the theme of performance throughout, the exhibition will include a significant grouping of artist-conceived instruments, scores for both music and events, as well as photographic and moving-image documentation. Artists' widespread use of technology surfaces as a dominant theme as well—from film, video, and other means of recording and playback to early experiments with computing and programmed displays of light and motion. Other parts of the exhibition will consider the influx of information and systems in early conceptual art, as well as the evolving theme of participation as artists increasingly relied on the viewer for active input and even a hand in making.
The Walker's renowned collection of Fluxus works—consisting of hundreds of event scores, editioned multiples, and packaged oddities—will serve as a through line across the various themes of the exhibition. In the late 1950s and early 1960s, an international constellation of artists, writers, performers, and composers assembled in performance halls and alternative spaces in the United States and Europe to challenge conventions through their freewheeling embrace of art in everyday life. In 1989, at the time of its landmark acquisition of some 400 Fluxus objects, the Walker became an early champion of this important facet of the international avant-garde. Now, some 50 years after the emergence of Fluxus and 25 years since the Walker's milestone acquisition, Art Expanded, 1958–1978 presents this pivotal work for reexamination and reinterpretation—this time within the broader context of the period's innovations and its unruly spirit of artistic reinvention.
Exhibition highlights include:
-Key paintings by Jasper Johns, Yoko Ono, Piero Manzoni, Daniel Buren, Niki de Saint Phalle, Jack Whitten, Alan Shields, and many others.
-An expansive display of Fluxus multiples and ephemera, as well as a focused presentation of Fluxfilms by Nam June Paik, Yoko Ono, Paul Sharits, and George Maciunas.
-A unique presentation of archival materials and never-before-seen photographs documenting Mushroom, a 1962 "happening" conceived by Allan Kaprow for the Lehmann Mushroom Caves in St. Paul, Minnesota.
-Belgian poet, filmmaker, and conceptualist Marcel Broodthaer's slide projection piece, Bateau Tableau (1973), in which the artist rigorously deconstructs a found nautical oil painting into successive close-up images.
-A dedicated screening space with films from the Walker's own Ruben/Bentson Film/Video Study Collection, including works by Merce Cunningham, Carolee Schneemann, Yvonne Rainer, and other pivotal figures who bridged the visual and performing arts of the day.
-An immersive installation of newly conserved 1960s light and kinetic artworks associated with the Walker's pioneering 1967 exhibition Light/Motion/Space
-A projection space dedicated to the Walker's recently acquired archive of films by queer performance artist and underground filmmaker Jack Smith.
Art Expanded, 1958–1978 is organized by the Walker Art Center and curated by Eric Crosby.
Art Expanded, 1958-1978
Opening Night: Northern Spark
Saturday, June 14 FREE
The lights stay on and the galleries remain open for Northern Spark, the annual city-wide dusk-to-dawn festival. Join us for a range of indoor and outdoor activities to engage curious all-night adventurers. The Garden Café by D'Amico and a range of food trucks stationed next to Open Field—AZ Canteen, A Cupcake Social, and Gastro Truck—provide fuel throughout the night. Northern Spark is presented by Northern Lights.mn, a nonprofit arts organization whose mission is to transform our sense of what's possible in public space.
Alison Knowles: Make a Salad
Thursday, July 10, 5–9 pm FREE
Open Field
Alison Knowles, a leading member of the Fluxus artist group, will be in residence with her collaborator, Joshua Selman, to re-stage her event score Make a Salad on Open Field. The performance includes Knowles preparing a massive salad by chopping ingredients to live music, tossing it in the air, and serving it to the audience. Originally performed in London at the Institute of Contemporary Art in 1962, Make a Salad has been re-staged more than a dozen times in cities around the world, most recently at the Highline in New York City in 2012, and in London at Tate Modern in 2008. Alison Knowles' work is included in the exhibition Art Expanded: 1958-1978.
Gallery Talk: Eric Crosby on the Expanded Arts
Thursday, July 31, 7 pm FREE
Galleries 1, 2, 3 and Perlman
Join Eric Crosby, curator of the exhibition Art Expanded, 1958–1978 for a conversation about the ways artists of the period—discontent working within the parameters of conventional artistic mediums—forged radically new hybrid forms.
Major support for the exhibition is provided by the Bentson Foundation. Additional support is generously provided by Mike and Elizabeth Sweeney.


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Germany, Czech Republic, Slovakia, Hungary..
June 6, 2006, 500 miles (805 km) - Total so far: 1,150 miles (1,851 km)
Well, would you believe I have actually made it to Budapest! I guess I only have to ride a few miles out of town and will have broken my record from the last attempt, hooray! I seem to be flying along now, helped by a splendid tailwind for the last week, though this also bought rain and cold which isn't too nice. Having a great time still and meeting loads of great people, the legs are slowly getting stronger, which is good news so hopefully will be able to get up some of those mountains that are to come.


text: M060124808 - Grade By Grade Flute Grade 1: The complete resource for the Grade 1 flautist. The Grade By Grade series draws on the rich and varied Boosey and Hawkes catalogue of classical, contemporary and educational repertoire, highlighting composers including Serge Prokofieff, Dmitri Shostakovich, Karl Jenkins, Carol Barratt and Christopher Norton, alongside arrangements of traditional music fro m around the world by Peter Wastall, Vera Gray and others. Packaged with a CD.


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You've all seen Ashley's styling work here (and I've also had her on the blog a million other times – she's just that chic) so I've asked her help for puting together three looks to ring in the new year. Most of these are already items you probably have in your closet. Take it away, Ashley!
This outfit is just a blazer and denim combo. I chose a pair of black high waisted denim because both of those attributes make for a more sophisticated look. Throw on your favorite slinky tee shirt and top with a fancy blazer or jacket. This particular one is velvet with gold threading making it super shiny. Add a heel, but make sure it has a little weight to it. Dainty is not the look we are going for. Top it off with your favorite accessories (a statement necklace is almost always a good choice) and you are done!
Get ready to dance the night away in this outfit. Whether it is at a friend's house or at a club, this outfit will work! A glittery or sparkly top, tunic, or dress will fit the bill. I chose a beaded, long sleeve tunic. Since it is a tunic with pretty high slits up the side, I wore a pair of brown leather shorts underneath. If you chose to go the tunic route, this is a great option. You are covered to dance your heart out but since the shorts and tunic should hit at about the same place, it gives the illusion of a super short sexy dress! I added a pop of color with a fun lace up wedge (easier for dancing in) and a vintage beaded bag. To make it even more special, I used a necklace to make a cool flapper style headband. And don't forget the tights! A cold girl is not a sexy girl.
The Look: Fancy Pants
For the final look, I made sure it was black tie ready. You never know where you might end up starting 2013. This is another beaded dress but with a completely different feel. A floor length, black dress with a sexy back usually will never do you wrong. I added a silver belt to give a little definition at the waist. With this neckline I didn't want to take away from it with jewelry, so the belt also works as a little shine in place of a necklace. This outfit is as easy as it comes. Just a dress, a belt, and shoes. If you change up the scene to something a bit more casual or want to just rock a fancy dress whoever you go, throw on a long cardigan. And if it's the latter, you should probably pair it with your favorite chucks!
Thanks so much Ash! You can get more styling inspiration from Ashley on her blog Sweet William.


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UEFI Install Guide
This tutorial will show you how to install Funtoo on a UEFI system. UEFI, also known as the Unified Extensible Firmware Interface, is a new firmware interface that is used on some newer computers as a replacement for the traditional PC BIOS. It has an integrated boot loader, so setting up booting is different. The recommended approach is to follow the Efi Stub Method . Many have reported that they are now unable to boot their system using the other, older method.
This tutorial is meant to be an "overlay" over the Regular Funtoo Installation. Follow the normal installation and only follow steps in this tutorial when dealing with partitioning and configuring the boot loader (GRUB). All steps are otherwise identical to the regular installation process.
What Are We Doing?
This guide will show you how to set up your UEFI system to load the GRUB boot loader, which will then load your Funtoo Linux kernel and initramfs. This is the "UEFI + GRUB" method as described on the Boot Methods page.
First Steps
To install Funtoo Linux on a UEFI system, first you need to boot SysRescueCD in UEFI mode. To do this, enable UEFI in your BIOS, and if necessary disable legacy booting. After some fiddling, you should be able to boot SysRescueCD and get a black and white text menu instead of the traditional aqua/cyan-colored menu. The black and white menu indicates that you booted SysRescueCD in UEFI mode. Once you've accomplished this, you're ready to continue with your Funtoo Linux installation and partition your drive. See below for details.
If the /sys/firmware/efi directory exists, then you have successfully booted in EFI mode and will be able to configure your Funtoo system to boot in EFI mode. If the directory doesn't exist, fix this first. It is a requirement for setting up EFI booting.
To set up your partitions for UEFI booting, you will create a ~500MB FAT32 partition on /dev/sda1, and set it to type EF00 using gdisk.
Command: n ↵ Partition Number: 1 ↵ First sector: ↵ Last sector: +500M ↵ Hex Code: EF00
This partition will serve as your Funtoo /boot filesystem as well as the partition that the UEFI firmware can read to load GRUB. Then you will set up swap on /dev/sda2 and your root filesystem on /dev/sda3. To create the FAT32 filesystem, type:
root # mkfs.vfat -F 32 /dev/sda1
Your /etc/fstab entry for this filesystem will also differ, and will look like this:
/dev/sda1 /boot vfat noatime 1 2
Make sure you add VFAT support to your kernel if you are building it manually.
EFI Framebuffer
If you have the following option enabled in your kernel, then uvesafb and efifb will not be able to detect the framebuffer:
Under Bus options (PCI etc.):
[*] Mark VGA/VBE/EFI FB as generic system framebuffer (NEW)
If you have that option enabled, you must also enable:
Under Device Drivers-->Graphics support-->Frame buffer Devices:
[*] Simple framebuffer support
This is the preferred method of using the EFI framebuffer, the efifb and uvesafb drivers will be used as a fallback if the above is not compatible.
Grub method
Unmask Grub 2.02_beta2
Unmask the latest version of GRUB by placing this in your
The 2.00 version of GRUB has known issues with UEFI booting. Using 2.02 is essential for having this boot method work reliably.
Emerging GRUB
You will still use GRUB as a boot loader, but before emerging grub, you will need to enable EFI booting. To do this, add the following line to /etc/portage/make.conf:
Then, emerge grub. You will notice efibootmgr getting pulled in as a dependency. This is expected and good.
Installing GRUB
Now, for the magic of getting everything in place for booting. You should copy your kernel and initramfs (if you have one -- you will if you are following the default install) to /boot. GRUB will boot those. But how do we get UEFI to boot GRUB? Well, we need to run the following command:
root # grub-install --target=x86_64-efi --efi-directory=/boot --bootloader-id="Funtoo Linux [GRUB]" --recheck /dev/sda
This command will simply install all the stuff to /boot/EFI and /boot/grub that your system needs to boot. In particular, the /boot/EFI/grub/grubx64.efi file will be created. This is the GRUB boot image that UEFI will load and start.
A more detailed explanation of the flags used in the above command:
--target=x86_64-efi: Tells GRUB that we want to install it in a way that allows it to boot in UEFI
--efi-directory=/boot: All GRUB UEFI files will be installed in /boot
--bootloader-id="Funtoo Linux [GRUB]": This flag is not necessary for GRUB to boot. However, it allows you to change the text of the boot option in the UEFI BIOS. The stuff in the quotes can be set to anything that you would like.
--recheck: If a device map already exists on the disk or partition that GRUB is being installed on, it will be removed.
/dev/sda:The device that we are installing GRUB on.
Configuring GRUB
OK, now UEFI has the GRUB image it needs to boot. But we still need to configure GRUB itself so it finds and boots your kernel and initramfs. This is done by performing the following steps. Since boot-update doesn't yet support UEFI, we will use boot-update, but then edit our
/boot/grub/grub.cfg to support UEFI booting.
First, you will need to edit
/etc/boot.conf. Format this as you would if you were booting without UEFI. If you are not sure how this should look, below is an example of what it could look like if you are booting from an unencrypted ext4 partition:
boot {
generate grub
default "Funtoo Linux"
timeout 3
"Funtoo Linux" {
kernel vmlinuz[-v]
params += rootfstype=ext4 root=/dev/sda2
After you have edited your
/etc/boot.conf file, run
boot-update. If you check your
/boot/grub/grub.cfg now, you should see something like this:
set timeout=3
insmod part_gpt
insmod fat
set root=(hostdisk//dev/sda,gpt1)
search --no-floppy --fs-uuid --set 3CFD-6884
if loadfont /grub/unifont.pf2; then
set gfxmode=text
insmod gfxterm
insmod vbe
terminal_output gfxterm
set menu_color_normal=cyan/blue
set menu_color_highlight=blue/cyan
menuentry "Funtoo Linux - vmlinuz-3.16.3" {
insmod part_gpt
insmod fat
set root=(hostdisk//dev/sda,gpt1)
search --no-floppy --fs-uuid --set 3CFD-6884
linux /vmlinuz-3.16.3 video=uvesafb:1920x1080-32,mtrr:3,ywrap rootfstype=ext4 root=/dev/sda2
set gfxpayload=text
set default=0
To get your
/boot/grub/grub.cfg to support booting with UEFI, make your
/boot/grub/grub.cfg look like this:
set timeout=3
insmod part_gpt
insmod fat
insmod efi_gop
insmod efi_uga
set root=(hostdisk//dev/sda,gpt1)
search --no-floppy --fs-uuid --set 3CFD-6884
if loadfont /grub/unifont.pf2; then
set gfxmode=auto
insmod gfxterm
insmod vbe
terminal_output gfxterm
set menu_color_normal=cyan/blue
set menu_color_highlight=blue/cyan
menuentry "Funtoo Linux - vmlinuz-3.16.3" {
insmod part_gpt
insmod fat
set root=(hostdisk//dev/sda,gpt1)
search --no-floppy --fs-uuid --set 3CFD-6884
linux /vmlinuz-3.16.3 video=uvesafb:1920x1080-32,mtrr:3,ywrap rootfstype=ext4 root=/dev/sda2
set gfxpayload=keep
set default=0
The lines that we have added and altered do the following:
insmod efi_gopand
insmod efi_uga: Both of these involve adding support for the UEFI framebuffer to GRUB.
set gfxmode=auto: Instead of having the GRUB boot option screen being displayed at the smallest resolution possible, changing this to auto will make it fit the resolution of your display.
Known Issues
- With pure UEFI boot mode, with legacy mode disabled, following error expected:
- video driver not supported, boot hangs, hard reboot required.
- Choose UEFI first, next legacy driver. It depends on motherboard vendor and efi bios version.
- In UEFI bios choose grub option, if your succeeded with above guide, additional menu should appear in Boot Menu, otherwise it boots into EFI shell:
grub:NAME of you hard drive
- In UEFI bios choose grub option, if your succeeded with above guide, additional menu should appear in Boot Menu, otherwise it boots into EFI shell:
- On some systems, installing the packages that are required for UEFI booting with any gcc later than a 4.x.x release may lead to a black screen after the GRUB screen. To fix this, before you begin installing any packages on your system, emerge =gcc-4.6.4-r2 and proceed with the installation as usual. Remember to switch your compiler back to the version of gcc that came with your system after you have finished installing. To do this, use
gcc-config 2. This problem can also be fixed by following the Efi Stub guide instead of the GRUB one.
Remember to follow all other steps in the regular Funtoo Install Guide. Assuming you did everything correctly, your system should now boot via UEFI! We will be adding UEFI support to boot-update soon to make this process easier.


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The core of the house heating system is its "Solar Heat Pump" which uses a heat pump water heater (HPWH) to provide domestic hot water, space heating and supplemental space cooling. The solar heat pump uses waste heat harvested from beneath the photovoltaic array to enhance the heat pump water heaters heating efficiency. Removing the warm air from beneath the PV array may also increase the efficiency of the PV array. To optimize daily energy collected, the heat pump operates when the air under the solar collector is at its warmest. The energy collected is stored as hot water and as heat in non-passive solar heated portions of the concrete slab floor.
The system draws warmed air from under a solar electric array and moves it across the evaporator coil of an air-to-water heat pump. This enhances the performance of both the heat pump and the solar electric array. Hot water is then stored and used for both domestic and space conditioning. We explored three heat pump configurations, three solar array configurations, and two control strategies. All system configurations have the following common elements:
- A solar plenum
- An air-to-water heat pump
- Energy storage
- Controls
Figure 1 shows a simplified air and water flow for the system. Airflow to the heat pump evaporator can come from under the solar array and be exhausted, or it can be drawn from occupied space and returned to occupied space as cool air. None of the designs considered would bring air from the solar plenum into the occupied space.
During the cooling season, the heat pump can be used to provide cooling to the space while heating water for domestic use. The warmest air inside the home can be drawn through the heat pump evaporator, cooled and delivered back into the space. This will provide roughly 48,000 Btu of cooling per day, which is sufficient to meet the design cooling loads in Oregon, while at the same time heating water for domestic use.
Solar photovoltaic electric power generation is a proven and reliable power source with great reductions in cost presently occurring. Photovoltaic power systems currently provide power for over a million structures worldwide. The application of photovoltaic power has traditionally not been applied to heating and cooling systems because of the intense electrical loads required by the equipment. Historically photovoltaics have been used for lighting loads in residences or commercial buildings. This project is an effort to demonstrate the potential of photovoltaics for heating and cooling equipment.
The potential for heating and cooling photovoltaic applications has been greatly enhanced by the integration of the ground source heat pump and the use of thermal storage. These two elements of mechanical system integration, ground source heat pump and thermal storage, are required to bring the economics of a photovoltaic powered heating and cooling system into feasibility.
The solar assisted heat pump provides an operating coefficient of performance from 2 to 8 times that of direct electrical power. A 100 watt photovoltaic panel can only power a 100 watt light bulb whereas a 100 watt photovoltaic panel can provide 400 watts of heat from a heat pump with a coefficient of performance of four.
The thermal storage system allows energy to be stored in water. In this system an existing four ton gas/electric heating and cooling system is being replaced with a 1 ton heat pump because the energy is being stored in water tanks. The water is then circulated across a fan-coil to provide the load. The reduction in peak system size makes the concept of photovoltaic power appropriate for heating and cooling system. Not only is the mechanical system size reduced in size to 1 tons but the required capacity of photovoltaic panels is reduced by the same amount.
The system is a grid tied system to take advantage of the electrical storage capacity of the grid. During start-up of motors and fans in the system a starting current of as much as four times the run current is required. If the system were not grid tied, the photovoltaic panels would be required to be sized that much larger to provide starting capacity.
The purpose of the system grid-tie capability is not for selling power back to the utility. The power created by the photovoltaic panels must be used for heating and cooling to take advantage of the COP.
Thermal Storage Water Tank
The concept of storing energy (thermal storage) as hot or cold water or as ice is already commonly used in many industrial and commercial building systems. Many commercial and industrial buildings use thermal storage as a way to offset utility rates off a peak period and allow the equipment to run more efficiently. Typically in these systems water surrounding refrigerant tubing in large tanks is frozen to store the cold thermal energy over night. This cold ice water is then circulated through the building or cooling process during the high load peaks of the day.
Energy is saved in two ways in these types of systems. Electrical power costs required to operate the system is lower at off-peak times. Mechanical equipment runs more efficiently at lower temperatures such as the night when ambient temperatures have dropped greatly.
In this mechanical system, one 120 gallon and one 80 gallon hot water tank is used for storage. The 120 gallon maximum size was driven by the code requirement that any tank over 120 gallons must be ASME rated greatly increasing the cost.
The design solution for thermal storage emerged around the use of common electric water heater tanks. The most cost-effective configuration would use either 80-gallon or 120-gallon sizes. The 80-gallon electric water storage tanks with 12-year warranties have a retail cost of around $350. The 120-gallon tanks cost nearly twice as much. Tank sizes large than this required additional testing. In addition, because they are manufactured in much smaller quantities, they are consequently more expensive. To build a storage system larger than about 240 gallons would result in substantially increased floor space and standby losses.
At 150 F, our two tank storage system (~200 gallons of water) is capable of providing 100,000 Btu of useful energy for space heating. Thus, if our heat pump is capable of making 150 F during the daytime, we could avoid running the heat pump water heater during the night, when its COP is lowest.
Figure 4 shows final storage configuration for heat pump Option B. A tempering valve is shown on the output of the backup tank to prevent scalding. A small electric backup tank (20 to 30 gallons) is needed because the space heating system may draw the water temperature down below 100 F, which is too cool for domestic hot water.
How much storage is sufficient to meet 80 to 95 percent of the demand and to prevent the heat pump from running during the coldest part of the day? The answer depends largely on the homes domestic hot water and space heating loads, the maximum temperature the heat pump can heat to, and how important it is to only collect heat during the daytime when solar pre-heated air can be fed to the heat pump.
To simplify the design, we have assumed that such a system would be installed in very well-insulated home with a net effective UA value of around 260 Btuh/ft2-F. The heat pump operating characteristics will determine the minimum operating temperature and the maximum stored water temperature.
If energy efficiency was our only consideration, water should be heated to the lowest possible temperature storage, thereby reducing losses, and keeping the heat pump from having to make water at higher temperatures where its COP is low. This is impractical, however, as it would limit the space heating system to low-temperature radiant floor systems or require the use of very large storage systems.
To get a general sense of how much storage is needed, consider how much energy is needed on a 90 percent design condition day in a 5000 HDD climate. Domestic hot water heating amounts to about 40,000 Btu per day. Daytime space heating loads are about 70,000 Btu and nighttime space heating loads are about 150,000 Btu. Thus, we would need approximately 260,000 Btu/day for this well-insulated light frame home. During the daytime, the heat pump will need to provide all space heating and water heating needs and still be able to charge the water storage tank to full temperature.
Space-heating energy can be delivered to the home in a variety of different ways. Energy stored as hot water may be delivered to rooms through the use of radiant floor circuits, individual fan-coils, hydronic baseboard units, a central air handler and hydronic coil, or any combination of these. Because of the net energy goal, however, the amount of electricity used by and losses in the delivery system(s) have to be carefully considered.
The Rose House uses two modes of heat delivery:
- An "energy recovery ventilator
- A radiant floor heating system
An energy recovery ventilator is a high efficiency heat transfer device that allows the transfer of the warm air exhausted from the house to the cool air coming from outside the house. The Stirling unit is so efficient that it can transfer nearly 90% of this heat.
The house is so tightly constructed against air leakage that it is necessary to draw outside air in to the house. This outside air is then preheated by the ERV. A hot water heating coil heats the air further on the way to the house when it is required.
After careful review of the current ERV models, a Stirling 200DX was chosen for this system. The 200DX model can use variable speed fan motors to deliver between 70 and 230 cfm of air. This ERV has several features that make it the best choice for this work:
- Energy recovery efficiency is above 90 percent at low speed and above 70 percent at high fan speed;
- Three ECM motors are used for fans and the heat exchange wheel allowing it to draw only 43 watts to deliver 70 cfm in low speed;
- A variable air volume can be easily set up by using the 0-10 vdc inputs to the fan speed motor control;
- The unit can be placed in "night-flush" mode. This is a night-time cooling mode where built-in sensors and software manage the economizer function.
The ERVs "night-flush" is unique among HRVs (Heat Recovery Ventilation) and ERVs cooling requirements. This is accomplished by stopping the heat transfer wheel from spinning and allowing air to be drawn into the house and exhausted without heat transfer. The ERV automatically monitors indoor and outdoor temperatures to prevent air from being drawn into the house unless it is cooler than indoor air. The night-flush mode stops once the interior temperature reaches 62 F.
To space heat with the ERV, a hydronic coil is added downstream of the ERV supply fan and a pump to respond to thermostat calls for space heating (or cooling). Several different companies are capable of providing small area three or four-row coils for less than $200. These specify coils that deliver more than 10,000 Btu/hour at 200 cfm and 120F entering water temperature at 5 gpm, while imposing an external static pressure penalty of less than 0.1 inch w.g. on the air side.
A limiting factor with this ERV/coil approach is ensuring that the delivered air temperature is warm enough for occupant comfort yet use the lowest possible water temperature. The big concern for this system design is in the morning when heat is being drawn for space heating, that the water is warm enough to meet shower needs.
Backup Heating System
Because of the significant downsizing of house loads and the systems used to meet them, 100 percent design temperature conditions, especially in colder climates, are a much larger challenge to system capabilities. For now, we generally see some rather conventional backup heating strategies to meet the last increment of space heating demand as being essential.
At a minimum, the electric resistance elements in the hot water storage tanks will serve this function. Some households will use strategically placed zonal electric units, wood heat, or a natural gas-fired appliance for backup heat.