Difference between revisions of "Talk:Group 9 Project - Fluorescent Proteins"

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Hey guys shaib i can't talk about cloning gfp as vishnu's doing it.--[[User:Z3254934|Shoahaib Karimi]] 04:37, 4 May 2010 (UTC)

Revision as of 15:37, 4 May 2010

I guess you can take a few notes on disadvantages, i'll take some on advantages and Shoahaib can make some notes on intro. Then we discuss our findings on Tuesday. --Vishnnu Shanmugam 14:20, 1 May 2010 (UTC)


I'll definately be there from 12. Also, should I write up the disadvantages section or do you want to do the intro/advantages/disadvantges together so we can each contribute things we've picked up from our sections? I've put what i've done so far down the bottom, but it's still a work in process. --Louisa Frew 12:10, 1 May 2010 (UTC)

Hey gang, so the plan is everyone have their written material posted up by Tuesday. We then meet on Tuesday (12pm-3pm) at cell bio lab for a discussion on where we go from there. Remember to bring your image collection on Tuesday. --Vishnnu Shanmugam 02:27, 30 April 2010 (UTC)


Sounds fine to me Louisa. Good idea dividing the different fluorescent proteins based on their emission spectra. "What makes a good fluorescent protein" could come under the "advantages" section, this will allow you to describe what good properties the FP has and why this is an advantage. Photoactivatable FPs and the development of infra-red FPs sounds good. See ya at lab, we discuss this more. --Vishnnu Shanmugam 08:55, 27 April 2010 (UTC)


Hi Guys. I've had a look how i'm going to do the "new FP" section, and i have a few different options which i thought i'd run past you. It's looking like the best way to approach it is to look at the variants of GFP and split them into Yellow, Blue, Orange and Rec spectrum categories to discuss the properties, advantages/disadvantages and uses of each. Does this sound like an ok approach? This will probably also need some info on what makes a "good" fluorescent protein- brightness, photostability etc. Do you think this would fit under any of the existing headings?? I have a couple of really good review articles that look specifically at this stuff so it shouldn't be very hard. Also, apart from colour variants there are lots of other types of FPs so i might just focus on photoactivatable FPs and the development of infra-red FPs if that sounds ok?!? Anyway, if you don't get this, then I'll catch you both in the lab tomorrow. --Louisa Frew 03:58, 27 April 2010 (UTC)

Hi! I had a look and completely agree we were a little over enthusiastic with the number of subtopics! Given the changes, we should defintately sort out a new distribution of sections in the lab today so we can get properly started. --Louisa Frew 01:57, 31 March 2010 (UTC)

Hi folks, I had a look at some of last year's cell biology group projects (can be found on the 2009 student link after clicking on 2010 projects). I propose that we consider revising some of our subtopics as they seem to cover the same thing. Eg. 3)Early/Historical Uses of GFP and 4)History development (GFP). Another is 6)Contemporary/commercially available fluorescent proteins and 7)Uses in current research which once again is the same thing as it is not possible to write about current research without writing about Contemporary/commercially available fluorescent proteins. We probably don't need this many topics but make each one detailed. Example:

  1. Introduction------S
  2. History of fluorescent proteins- this includes all GFP (development,nobel prize, historical uses)-------S
  3. Development of new fluorescent proteins - new proteins since GFP and how they were developed---write detailed about most important fluorescent proteins-----L
  4. Current research - How the new fluorescent proteins are used and to what purpose in research projects today eg. nuclear staining-------V
  5. Advantages of fluorescent proteins-------V
  6. Limitations of fluorescent proteins-------L
  7. References
  8. Links to current research sources (people and organisations)
  9. Glossary

Have a look at the 2009 projects and tell me what you guys think. --Vishnnu Shanmugam 01:31, 30 March 2010 (UTC)


Project outline: (please add suggestions)

  1. Introduction- define fluorescent proteins
  2. The fluorescence process- general explanation L
  3. History- development (GFP)- Nobel Prize etc S
  4. Early/Historical Uses of GFP L
  5. Developments that followed GFP- new proteins (colours, variations etc) S
  6. Contemporary/commercially available fluorescent proteins- different brands, uses etc V
  7. Uses in current research V
  8. Future ??
  9. References
  10. Links to current research sources (people and organisations)
  11. Glossary

Some Subtopics for Fluorescence techniques:

  1. Immunofluorescence
  2. Fluorescence in situ hybridization
  3. Fluorescence Microscopy
  4. Fluorescence trangenesis
  5. Flow Cytometric Fluorescence
  6. Fluorescent marking and labelling
  7. Fluorescent Proteins
  8. Fluorophores

Hello! Fluorescence is still looking like a good option. We've added a few to the list! See you next week. --Louisa Frew 07:00, 17 March 2010 (UTC)3 .



hey i found great articles but 1 of them is german i have an exam soon so ill get on to translating it.




Vishnnu's Research

Cancer

Cancer (malignant neoplasm) is a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion of surrounding tissues and metastasis (spread via the circulatory or lymphatic system). The first use of GFP to visualize cancer cells in vivo was by Chishima et al. They stably transfected tumour cells with GFP and transplanted these into several mouse models, including orthotopic models that have a high metastatic capacity. They showed that in excised live tissue, with no additional preparation, metastases could be observed in any organ at the single-cell level. In addition, cells were visualized in the process of intravasation and extravasation. The visualization of single metastatic cells in tissue is beyond the capabilities of standard histological techniques and so such ex vivo studies enabled, for the first time, micrometastases (including dormant cells) to be visualized in unfixed or unprocessed tissue.

Researchers can attach the fluorescent molecules to a protein inside a dividing cancer cell, then by shining a light of the appropriate colour, scientists can watch as a cell divides uncontrollably. Fluorescent proteins (FPs), because of their endogenous expression, allow the observation with minimal disturbance to the subject (Hoffman and Yang, 2006). For example, cancer cells can be engineered to carry FPs stably and implanted into the subject to allow monitoring of metastasis and the effectives of cancer treatment.

Previously developed fluorescent compounds that are activated inside the body's cells have the limitation that, once they are turned on, they continue to fluoresce even after they diffuse to new locations, making it difficult to distinguish viable tumor cells from normal tissue or dead or damaged tumor cells. The research team, led by Hisataka Kobayashi at the Molecular Imaging Program of NCI's Center for Cancer Research (CCR), in collaboration with Yasuteru Urano at the University of Tokyo, created an imaging compound that is turned on only when it is inside a living cell and stops fluorescing when it leaves the cell, as would happen when the cell dies or becomes damaged. The compound also can be engineered to target specific types of cancer cells.

Fluorescent imaging based on the specific marking of tumors is widely used in experimental oncology. The possibility to introduce genes of a particular class of fluorophores [fluorescent proteins (FPs)] into cells enabled the development of a new method: genetic marking. The fluorescence ability of FPs persists for the whole life of a cancer cell and remains after cell division. As a result, it becomes possible to estimate tumor growth rate, to study the mechanism of carcinogensis and metastasis formation, and to investigate the safety and efficacy of intervention using novel therapeutics. Recently, a new group of FPs - red fluorescent proteins (RFPs) - was isolated, and they became useful as markers for whole-body biological imaging. The fluorescence spectrum of these proteins is in the relatively long-wave part of the spectrum (580 to 650 nm), a region that is promising for object visualization at depths up to 1 to 2 cm with millimeter resolution. Therefore, RFP-labeled tumors can be regarded as the most appropriate model for whole-body investigations.


Malaria

Malaria is caused by a parasite called Plasmodium, which is transmitted via the bites of infected mosquitoes. In the human body, the parasites multiply in the liver, and then infect red blood cells. The disease is caused by infection with one of four species of the genus Plasmodium: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. The first two are the most common.

Malaria is one of the most widespread of all human parasitic diseases, and in the early part of the last half century more than two-thirds of the world's population lived in endemic areas. WHO estimates that 3.3 billion people (half of the world's population) are at risk of malaria. Every year, this leads to about 250 million malaria cases and nearly one million deaths. People living in the poorest countries are the most vulnerable. Malaria is especially a serious problem in Africa, where one in every five (20%) childhood deaths is due to the effects of the disease. An African child has on average between 1.6 and 5.4 episodes of malaria fever each year. And every 30 seconds a child dies from malaria. In Australia, malaria has been endemic, but was declared eradicated from the country in 1981. Although malaria is no longer endemic in Australia, approx. 700-800 cases occur here each year in travellers infected elsewhere, and the region of northern Australia above 19oS latitude is the receptive zone for malaria transmission.

A possible breakthrough in curtailing the spread of malaria carrying mosquitoes was reported in October 2005 the creation of mosquitoes with green fluorescent testicles. Now male mosquito larvae can easily be separated from female mosquito larvae. Without green fluorescent gonads it is impossible to separate mosquito larvae based on their sex. Now a laser sorting machine has been developed that can sort 180,000 larvae in 10 hours. Once separated from the females it is trivial to sterilize the males and release them into the environment where they will mate with wild females. Female mosquitoes only mate once in their two-week cycle, so if they chose a sterilized male they will produce no offspring. If a large enough population of sterilized males is released into the wild population should be eradicated in a fairly short time.


Prion

A prion is an infectious protein particle similar to a virus but lacking nucleic acid. An infectious prion can affect a normal prion protein. Whenever a prion comes in contact with a normal prion protein, it causes the normal protein to 'flip' into an abnormal shape, thereby becoming a prion (ie, the rogue form of the protein). Any other normal prion protein that a rogue prion touches will also be converted, creating a domino effect. Prions have been known to cause neuro generative diseases such as spongiform encephalopathy (mad cow disease and Creutzfeldt-Jakob disease), fatal familial insomnia and Gerstmann-Straussler-Scheinker Syndrome, chronic wasting disease and Scrapie.

Humans can be infected by two modes:

1. Acquired infection (diet and following medical procedures such as surgery, growth hormone injections, corneal transplants) i.e. infectious agent implicated.

2. Apparent hereditary mendelian transmission where it is an autosomal and dominant trait. This is not prima facie consistent with an infectious agent.


We need to know more about how PrPc is expressed and treated in cells in order to understand how the misfolding of PrPc occurs and why cells die as a result. By means of green fluorescent protein (GFP) cloned into PrP, PrP in cell cultures can be studied under a microscope. In addition, genetically manipulated variants of PrP have been made in order to uncover important factors regarding the localisation of PrP in cells and the enzymatic cutting of PrP.

PrP is normally cut into fragments in the course of its cellular lifespan. Lund et.al has studied one of these cutting processes, the α-cut. Where the PrP α-cut occurs in the cell, and to what purpose, is unknown. Through his studies, Lund has shown that PrP is cut in the same place, even when the amino acid composition at the place of cutting is changed. PrP is also cut at the same place, irrespective of whether it is joined to the outside of the cell membrane or whether it is localised in the cell cytoplasm. Lund's findings indicate that the cutting occurs at the same place in PrP, but that the cutting is caused by different mechanisms, depending on where the PrP is localised in the cell. A phenomenon associated with PrP's localisation in cells that is still poorly understood is that in some types of cells, PrP is positioned in the cell's cytoplasm instead of on the cell membrane, where it most likely fulfils its function. A predominant theory on why proteins may be found in the cytoplasm instead of on the cell membrane is that the cell in question is in a state of stress. Furthermore, PrP has been shown to have an inefficient signal sequence compared to other proteins and may therefore be less efficient at following its natural route out onto the cell membrane, even under normal cellular conditions. Lund's work reveals that a completely different mechanism related to the actual translation of PrP may also be the reason why a proportion of the PrP molecules end up in the cytoplasm. By studying different mutated variants of PrP, Lund has demonstrated that a cytoplasmic variant of PrP can emerge after PrP molecules have been synthetised from a downstream start codon in the PrP gene. The result of this translation is a shortened form of PrP which lacks large portions of the signal sequence and therefore ends up in the cytoplasm of the cell.

Research also focuses on how prions travel from the digestive tract and other sites in the body and pass into the brain? Scientists have found that organs involved in immune cell development and maturation—such as lymph nodes, spleen, and bone marrow—actually serve as the prions’ staging area, where they propagate. Subsequently, researchers speculate, peripheral nerves that stimulate these organs serve as conduits that transport prions to the spinal cord and brain. Investigators will test these hypotheses by using a novel technology that takes advantage of mice genetically engineered to produce prions that are fluorescent and will glow green under the microscope. First, researchers will analyze the distribution of fluorescent prions in the intestine, spleen, lymph nodes, and involved peripheral nerves. Next, they will analyze dissemination of prions within the spleen using a cell transplantation technique. Finally, they will study prions that are selectively generated in specific immune cells and nerve cells, to analyze the processes involved in transporting prions from the immune system to the brain. Significance: Learning how prions travel to the brain could lead to development of methods for blocking this process and preventing deadly prion infection in the brain.


GFP Variants Section- Louisa

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 (Shaner et al. 2007, Shaner et al. 2005).

Blue

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 (Shaner, 2007). New blue Aquorea GFP variants Azurite, SBFP2 and EBFP2 show improved brightness and photostability in comparison to EBFP, offering a promising means of imaging live cells in this region (Ai et al., 2007 & Kremers et al. 2007). EBFP2 is the most photostable and brightest blue FP (Shaner et al., 2007).

Cyan

The cyan FPs (CFPs) began with the production of enhanced CFP (ECFP) from Aequorea GFP (Cubbit et al. 1995). mCerulean followed in 2004 offering a brighter and better general-purpose CFP (Rizzo et al. 2004). In 2006, a monomeric teal-coloured variant mTFP1 was obtained from a Clavularia soft coral protein (Ai et al, 2006). mTFP1 is brighter, less pH sensitive and more photostable than the traditional CFPs, making it an excellent alternative to its predecessors (Shaner et al. 2007). 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 (Shaner et al. 2007).

GREEN 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 (Shaner 2007).

EGFP?????

Most of these novel GFPs exhibit no discernable advantage over EGFP (Shaner 2007) and hence will not be discussed. Emerald, a derivative of EGFP, is currently the best choice for live-cell imaging due to its more efficient folding than EGFP at 37°C (Shaner 2007. Cubitt 1999)

sfGFP????

Yellow

Yellow FPs (YFPs) are among the brightest and most versatile probes developed in any of the spectral classes (Shaner 2007). EYFP was developed in 1999 (Miyawaki et al, 1999) and is still widely used despite its high pKa and sensitivity to halides (Shaner et al 2007). EYFP has proved effective in tracking the distribution patterns of single proteins on the membranes of live cells (Ober et al. 2004). mCitrine, derived from the addition of a single mutation in EYFP, is less halide sensitive and twice as resistant to photobleaching as its predecessor (Griesbeck et al, 2001). 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 (Nagai et al., 2002). 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 (Nguyen and Daugherty, 2005).

Orange

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 (Shaner, 2007). 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 (Shaner, 2007). 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) (Karasawa et al. 2004). 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 (Shaner 2007 and Shaner 2004) 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 (Merzlyak et al 2007). Random mutagenesis of Tag RFP 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 (Campbell et al. 2002) 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 (Shaner et al 2004). The major drawback in the use of tdTomato is its comparatively large size which is proposed to interfere with fusion-protein packing (see reference 66 from the paper pp62).

Red

FPs that emit in the far red area of the spectrum are the holy grail of this area of development, due to this wavelength of light being less phototoxic and able to probe deeper into biological tissues (Shaner 2007). 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 (Shaner, 2007). Despite their improved emission colours, many Fruits lack the brightness and photostability needed for most experiments (Shaner 2005)). According to Shaner and team, mStrawberry and mCherry are the best reds, with brightness levels of 75% and 50% of EGFP (Shaner 2007). mCherry is more photostable than mStrawberry (Shaner 2005) and as well as a better alternative to mRFP1 for long-term imaging experiments (Shaner 2007).

Developed in 2004, mPlum is one of the first true far-red probes, emitting at 649nm (Wang et al 2004). 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 (Shaner 2004, Shaner 2007).

Kutushka, a dimeric protein that emits at 635nm, was developed in 2007 and is commercially available from Evrogen as TurboFP635 (Shcherbo et al 2007). 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 (Shcherbo et al. 2007). 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 (reference book).

Infa-red !!!!!!!!!!!

Other Novel FPs

Photoactivated FPs

These proteins display negligible fluorescence until excited by irradiation at a specific wavelength (Lippincott-Schwartz and Patterson 2003). 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. (EXAMPLES)

Photoconvertable FPs

This class of FPs show “light-driven modulation of fluorescence properties” (Wiedenmann et al, 2009 pp1032), allowing !!!!!!!!! Green-to-red photoconvertable 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!!!!!!!!!

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 (Li et al 1998)

"Fluorescent Timer" Protein

This protein, similarly to the destabilised GFP variants, allows the measurement of protein turnover and expression timing (Lippincott-Schwartz and Patterson 2003). It initially fluoresces in green area of the spectrum before conversion of the fluorophore after several hours leads to emission in the red (Terskikh et al.2000). Using the ratio of green to red fluorescence allows the age of the tagged protein to be determined.

Future aims for new mutants suggested by Shaner et al. : • Brighter • Monomeric • High contrast • Easily photoconverted • Reversible photoactivation • Red-to-green photoconversion • Improved expression in the far-red or near-infrared regions

--Louisa Frew 12:10, 1 May 2010 (UTC)




1565 N. Monardes, he observed emission of light by an infusion of wood lignum Nephriticum (first reported observation of fluorescence)

1640 Licetus, study of Bolognese stone. First definition as a non-thermal light emission

1842 E. Becquerel , observed emission of light by calcium sulfide upon excitation in the UV. First statement that the emitted light is of longer wavelength than the incident light

1853 G. G. Stokes Introduction of the term fluorescence

1858 E. Becquerel, created the first phosphoroscope

1929 F. Perrin Discussion on Jean Perrin’s diagram for the explanation of the delayed fluorescence by the intermediate passage through a metastable state First qualitative theory of fluorescence depolarization by resonance energy transfer

1944 Lewis and Kasha Triplet state

1955 Green fluorescent substance in jellyfish first described. (1)

1962 GFP identified as protein, extracted from 10,000 jellyfish - "a protein giving solutions that look slightly greenish in sunlight though only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite, has also been isolated from squeezates." Called "green protein." (2)

1979 Shimomura characterized structure of chromophore. (6)

1985 Prasher clones aequorin. (7)

1993 Structure of GFP chromophore confirmed, flanking amino acid residues corrected from Shimomura's 1979 structure. Still "apoGFP". (9)

2000 Fluorescent timer protein. (20) The biggest difference between green fluorescent protein and its red analog, DsRed, is that the chromophore of DsRed has an extra double bond (drawn in yellow) which extends the chromophores conjugation and causes the red-shift. (21) The Olympus web-site has a java tutorial that shows how the DsRed chromophore is formed. Check it out.

2002 Monomeric DsRed (mRFP) (22), first photoconverible (Kaede (23,24), and photoactivatable (PA-GFP (24)) FPs created.

2004 New "fruit" FPs generated by in vitro (26) and in vivo (27) directed evolution.










Hey guys shaib i can't talk about cloning gfp as vishnu's doing it.--Shoahaib Karimi 04:37, 4 May 2010 (UTC)