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Fluorescent Proteins Identification Lab

What is fluorescence? The process by which light is absorbed by a molecule and then re emitted at a longer wavelength, producing a particular color.
What is a fluorophore? the chemical structure in a fluorescent molecule that absorbs and emits the photons of light.
EGFP enhanced green fluorescent protein. has increased fluorescent light output. 35x more brightly. Ser64Thr
ECFP enhanced cyan fluorescent protein. changed the color from green to cyan.
EYFP enhanced yellow FP. changed color to yellow.
DsRed is from the coral Discosoma. It is a tetramer formed of 4 identical subunits of 26kDa.
The 2 parent proteins GFP and DsRed
FP genes were cloned into the plasmid vector... pRSET in the host bacterium E.Coli strain DH5alpha.
plasmids are circular double stranded DNA mol'cs capable of autonomous replication w/in host bacterium. they are not part of the bacterial chromosome.
vectors are a vehicle for maintaining and propagating cloned genes in a host organism, such as E.coli. foreign genes can by inserted into a plasmid vector and are rep'd along w/plasmid DNA inside bact. cell.
cloning the production of multiple identical copies of an organism, cell or gene. if a gene is isolated from genetic material of an organism, and inserted into a vector where identical copies can be rep'd, it is referred to as cloned.
PT7 bacteriophage T7 promotor. This sequence is the binding site for T7 RNA polymerase that will synthesize the mRNA.
RBS ribosome binding site. Sequence that binds the mRNA to the ribosome.
ATG vector start codon. This codon codes for methionine, and is the signal for the ribosome to initiate synthesis of the AA chain.
6xHis His-tag. This sequence encodes 6 histidine aa acids in tandem. These will bind to a nickel aff. column for fast and efficient purification of the protein encoded on the cloned gene.
Xpress Epitope Tag 8 aa sequence(DLYDDDDK). that is recognized by an antibody. This provides an alternative method for purifying(and detecting) the protein by aff. chromatography.
EK aa seq. that is cut by the protease enterokinase. it can be used to remove the preceding aa(met,6 his, DLYDDDDK) that are encoded by the vector.
MCS multiple cloning site. a series of tandem restr.enzy. cutting sites.(BAMHI-HindIII).this is where the gene encoding the protein of interest (DNA frag. containing pRSET and gene) is inserted into vector.
Stop vector translation stop. The stop codon TAA terminates translation from the mRNA.
f1 ori origin of rep. for f1 bacteriophage of e.coli. used in connection w/helper phage(which provides all necessary f1 gene products) to produce single str. copies of the plasmid w/cloned genes(useful for DNA of the cloned gene)
Ampicillin(BLA gene) the BLA gene that encodes beta-lactamase, the enzy. that degrades the antibiotic ampicillin. In the presence of ampicillin, only bact. containing a plasmid w/amp-resistance gene will live.
Ampicillin inhibits the enzymes that form peptide cross-links btwn carbohydrate chains and bacterial cell wall. This weakens the cell wall and lysis eventually occurs.
pUC an E.coli replication initiation site is inserted here. The DNA polymerases of the host bacterium bind to this site to replicate the vector DNA.
Where were the genes for FP's cloned to? The MCS of pRSET-B. BAMHI is the site cut by the restriction enzyme BAMHI, where the 5' end of the FP gene was insterted.EcoR1 is the site where the 3' end of the gene was inserted.
The role of glucose in Solution one of alkaline lysis mini plasmid prep? to increase the [solute] outside of the cell so when sol.II is added, osmotic pressure draws H2O out hopefully carrying the plasmid.
The role of EDTA in solution I of the alkaline lysis mini plasmid prep? EDTA is a chelator. It's main function is to remove Mg2+ and inactivate DNA nucleases for which this ion is cofactor.
The role of RNase in solution I of the alkaline lysis mini plasmid prep? It is a ribonuclease that will degrade contaminating bacterial RNA.
The role of NaOH in solution II? To raise the pH, which causes the DNA to denature and the separate strands will ppt from solution.
The role of 1% SDS Page in solution II? to lyse the cells by dissolving the cell membrane.
The role of Kacetate in solution III? It makes the solution more neutral. Causes plasmid DNA strands to re-anneal. K+ ions comples w/dodecyl sulfate ions and ppt.
Why doesn't the chromosomal DNA re-anneal in solution III? It is too big and complex to re-anneal under those conditions.
The role of Isopropanol? causes the plasmid DNA to ppt.
The role of ethanol wash? to remove remaining salts and detergents.
Purity of Plasmid DNA Is measured by the ratio of A260/A280. Greater than 1.6 is adequate. Greater than 1.8 is better. Protein contamination reduces the ratio.
Nucleotides absorb UV light with a maximum at? 260nm
The purpose of an alcohol precipitation is to? 1. concentrate DNA or RNA and/or 2. remove detergents, salts and other low MW contaminants. or to change buffer composition.
nucleic acids will not precipitate unless... there is a monovalent cation to neutralize the negative charges and permit DNA to aggregate.
NH4acetate is preferred when one wants to remove.. free deoxynucleotides, as they co-precipitate less with NH4 than Na.
LiCl is commonly used to ppt RNA, b/c it is more soluble in ethanol than Na, and efficient ppt of RNA requires large amounts of ethanol than DNA.
When ppting nucleic acids in the presence of SDS, why use K+? K+ is used when you want the SDS to ppt as well. SDS is soluble, and KDS or potassium dodecyl sulfate is is insoluble.
Why is water an excellent solvent for oppositely charged molecules? Since the polar water molecules orient in concentric shells around the charges, forming hydration spheres. This shields there charge so the attractive forces btwn them is less and they don't come together to form a ppt.
hydration sphere separates charges so the attractive forces btwn them is less and they don't come together and form a ppt.
When alcohol is added to solution, solubility is decreased for two reasons: 1.alcohols form Hbonds with water, which reduces the amt of water available to dissolve 2. the capacity of alcohols to shield opp charges and keep them apart is much less than water.
restriction enzymes are nucleases that are produced by bacteria to protect them from viruses that infect bacterial cells, called bacteriophage. They recognize a spec. seq of nucleotides in bacteriophage chrom. and produce a dbl str cut in DNA to prevent replication.
How does the bacterium protect its own DNA from being cut by restriction enzymes? By methylating adenosine or cytidine nucleotide in the sequence, which prevents endonuclease from binding.
How are restriction enzymes named? with the first letter of the genus, followed by the first 2 letters of the species, followed by the strain or serotype if any, and a roman numeral if the bacterium contains more than 1type of res. enzy.
Why does DNA have a uniformly negative charge? This is due to the acidic phosphodiaster groups.
Supercoiled DNA will migrate slower or faster than liner DNA on agarose gel electrophoresis? it migrates faster b/c it is more compact. plasmid dna is normally supercoiled
If DNA is nicked, how will this affect its migration down a gel? dna uncoils, but remains circular and usually migrates more slowly than linear dna of the same size
The 2 most common buffer systems for agarose gel electrophoresis are: Tris-acetate EDTA (TAE) and Tris-borate-EDTA (TBE).
What is the difference between using TAE and TBE as a buffer system? TAE gives a better resolution and is preferred when DNA is to be purified from the gel afterwards. TBE has a better buffering capacity.
What dye was used to track the proteins in the electrophoresis gel? bromophenol blue. it will run at about the same position as 300bp
Syber safe a fluorescent dye used in our electro gel to help visualize dna. it works by slipping btwn base pairs of dbl str. dna. it fluoresces at 500nm, and will detect down to 50ng of dna.
ethidium bromide a powerful mutagen. a fluorescent dye. fluoresces intensely at 560nm when excited by UV light (260-360nm), permitting detection of as little as 20ng dna.
competent cells cells that have been treated so they are temporarily able to take up DNA.
Method for making competent cells cold CaCl2 treatment of cells that are dividing rapidly, followed by heat shock.
What cell phase is transformation most successful in? early-mid log phase.
How is it believed that the CaCl2 allow dna molecules to cross through the plasma membrane? it is proposed that dna cross through adhesion zones where there are pores formed by the fusion of the inner and outer mbrn. adhesion zns only form in growing cells. Ca2+ shields - charge on dna and pm, allowing close contact,this may be favored by low T
how is it believed the heat shock helps with dna crossing through the PM? The heat shock could briefly destabilize the membrane so that somehow the dna is allowed to get across.
JM109-DE3 a strain of E.coli carrying the gene for T7 RNA polymerase.
T7RNA polymerase binds to the T7 promoter region in the pRSET-B plasmin and synthesizes mRNA coding for the regions marked in the pRSET-B plasmid map.
Why is JM109-DE3 preferred over BL21-DE3? JM109-DE3 grow slower, giving FP's time to mature to their full fluorescence.
2 controls for transformation 1. competent cells w/out plasmid: positive control 2. competent cells w/out plasmid on LB + ampicillin: negative control
What does the positive control tell us? (comp cells w/out plasmid) If nothing grows, then something may be wrong with competent cells.
What does the negative control tell us? (comp cells w/out plasmid + LB and ampicillin) tests to be sure ampicillin is still good (good for 2-3 months). Should see nothing.
3 pieces of evidence when determining the identity of the fluorescent protein. 1. Color of bacterial colony expressing the gene. 2. absorption spectrum 3. observing MW on SDS-PAGE gel.
His-Tag was added to which end of the FP? the N-terminal end.
NTA stands for nitrilotriacetic acid
NTA is a spacer molecule that nickel is chelated to.
the lysis and column wash buffers have a low concentration of free imidazole, what does this low concentration do? competes out low affinity binding to the resin of a single or two adjacent histidines in a protein.
What role does the high concentration of imidazole in the elution buffer perform? It is high enough to compete successfully for the nickel and displace the his-tagged proteins.
A molecule will only absorb light when... it contains a dipole that can be made to oscillate at the same frequency as the incident light. light sensing molecules in our retinas contain these dipoles.
following the absorption of photons, the 3 principle processes by which the excitation energy is dissipated or converted are: heat, chemical reactions and fluorescence.
Why didn't we use reducing agents when we ran our SDS-PAGE gels? to maintain fluorescence. prolonged exposure to SDS will also reduce fluorescence. This is why samples were saved in elution buffer not sample buffer.
what stain did we use on our SDS-PAGE gels? Coumassie blue
Where is the GFP fluorophore located in the AA sequence? Ser 65, Tyr 66, Gly 67
What issues had to be addressed in order to make FP's more useful in the laboratory? 1. Brightness of fluorescence 2. Color, the more colors, the more flexibility when planning experiments 3. Folding temperature,needed to be able to fold properly at warmer temperatures, at which bio systems and prtns of interest fcn 4. work as fusion prot
Where is the DsRed fluorophore located in the AA sequence? Gln 66, Tyr 67, Gly 68
Why is wild type DsRed problematic when labeling proteins of interest? It is a tetramer, and it is difficult to label a protein of interest with an FP that is more than one polypeptide.
What needed to be done in order to make DsRed useful? prevent the formation of a tetramer, and to obtain active fluorescence in the monomer form.
How many aa substitutions went into mRFP1? 33, but, from here, further substitutions produced variations in the color of the fluorescence.
Why was some of the sequence of GFP added to DsRed derivatives? after all the work to produce mRFP1, it did not work well as a fusion protein.
fusion protein the protein produced by combining coding regions of the genes of two different proteins in order to link them together as a continuous polypeptide.
Can things around the fluorophore also affect fluorescence? Yes, by changing the electrostatic environment around the rings.
To get wild type FP's to fold correctly, what aa substitution was made? Phe64Leu
Blue shift ECFP Tyr66Trp: Green to blue (shorter wave length emission max) Means emission to shorter wavelength
Red shift EYFP Ser65Gly shifts to a longer wavelength emission
Using a FP to label a protein of interest Use a mammalian expression vector and insert a fusion protein (inframe)
An example of FRET-Using a FP: Calcium binding protein fusion protein to detect Ca2+ influx. FRET needs 2 fluorophores.
FRET stands for Fluorescence Resonance Energy Transfer
bio luminescence in Jelly fish an enzyme called acquorin catalyzes a luminescent chemical rxn. the light given off is "blue light". If GFP is close to acquorin, RET occurs.
Created by: jriendea



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