Post on 12-Jan-2016
Green Fluorescent ProteinGreen Fluorescent Protein
Molecular GeneticsMolecular Genetics
Green Fluorescent ProteinGreen Fluorescent Protein
Green Fluorescent Protein (GFP) has existed for more than one hundred and sixty million years in one species of jellyfish, Aequorea victoria
Green Fluorescent Protein (GFP) has existed for more than one hundred and sixty million years in one species of jellyfish, Aequorea victoria
FluorescenceFluorescence
Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1, respectively. Its emission peak is at 509 nm in the lower green portion of the visible spectrum.
Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1, respectively. Its emission peak is at 509 nm in the lower green portion of the visible spectrum.
FluorophoreFluorophore
Eleven strands on the outside of cylinders form the walls of the structure. The cylinders have a diameter of 30A and a length of 40A.
Small sections of alpha-helix form caps on the ends of the cylinders and an irregular alpha-helical segment also provide a scaffold for the fluorophore which is located in the geometric center of the cylinder. The strands of beta-sheet are tightly fitted to each other like staves in a barrel.
Eleven strands on the outside of cylinders form the walls of the structure. The cylinders have a diameter of 30A and a length of 40A.
Small sections of alpha-helix form caps on the ends of the cylinders and an irregular alpha-helical segment also provide a scaffold for the fluorophore which is located in the geometric center of the cylinder. The strands of beta-sheet are tightly fitted to each other like staves in a barrel.
FluorophoreFluorophore The fluorophore itself is a p-
hydroxybenzylidene-imidazolidone. It consists of residues Ser65- dehydroTyr66 - Gly67 of the protein. The cyclized backbone of these residues forms the imidazolidone ring.
The fluorescence is not an intrinsic property of the Ser-Tyr-Gly tripeptide. The amino acid sequence Ser-Tyr-Gly can be found in a number of other proteins as well.
This peptide is neither cyclized in any of these, nor is the tyrosine oxidized. None of these proteins has the fluorescence of GFP.
The fluorophore itself is a p-hydroxybenzylidene-imidazolidone. It consists of residues Ser65- dehydroTyr66 - Gly67 of the protein. The cyclized backbone of these residues forms the imidazolidone ring.
The fluorescence is not an intrinsic property of the Ser-Tyr-Gly tripeptide. The amino acid sequence Ser-Tyr-Gly can be found in a number of other proteins as well.
This peptide is neither cyclized in any of these, nor is the tyrosine oxidized. None of these proteins has the fluorescence of GFP.
Absorption spectrum of gfp
Absorption spectrum of gfp
Excitation and EmissionExcitation and Emission
Amino acid Sequencegfp
Amino acid Sequencegfp
1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl
61 vttfsygvqc fsrypdhmkq hdffksampe gyvqertiff kddgnyktra evkfegdtlv
121 nrielkgidf kedgnilghk leynynshnv yimadkqkng ikvnfkirhn iedgsvqlad
181 hyqqntpigd gpvllpdnhy lstqsalskd pnekrdhmvl lefvtaagit hgmdelyk//
1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl
61 vttfsygvqc fsrypdhmkq hdffksampe gyvqertiff kddgnyktra evkfegdtlv
121 nrielkgidf kedgnilghk leynynshnv yimadkqkng ikvnfkirhn iedgsvqlad
181 hyqqntpigd gpvllpdnhy lstqsalskd pnekrdhmvl lefvtaagit hgmdelyk//
Blue Fluorescent ProteinBlue Fluorescent Protein
1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl
61 vttfxvqcfs rypdhmkrhd ffksampegy vqertiffkd dgnyktraev kfegdtlvnr
121 ielkgidfke dgnilghkle ynfnshnvyi madkqkngik vnfkirhnie dgsvqladhy
181 qqntpigdgp vllpdnhyls tqsalskdpn ekrdhmvlle fvtaagithg mdelyk
1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl
61 vttfxvqcfs rypdhmkrhd ffksampegy vqertiffkd dgnyktraev kfegdtlvnr
121 ielkgidfke dgnilghkle ynfnshnvyi madkqkngik vnfkirhnie dgsvqladhy
181 qqntpigdgp vllpdnhyls tqsalskdpn ekrdhmvlle fvtaagithg mdelyk
Blue Fluorescent ProteinBlue Fluorescent Protein
Blue fluorescent protein is a variant of the GFP with a Hit to Tyr substitution at position 66 and a second substitution from Tyr to Phe at position 145.
Blue fluorescent protein is a variant of the GFP with a Hit to Tyr substitution at position 66 and a second substitution from Tyr to Phe at position 145.
ChameleonsChameleons
Different mutations causes different colors
Different mutations causes different colors
Fluorescence in NatureFluorescence in Nature
Fluorescent Molecules used in research
Fluorescent Molecules used in research
Fluorescence in ResearchFluorescence in Research
DNA TransformationDNA Transformation
Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form
Competent cellcapable of taking up DNA
May be important route of genetic exchange in nature
Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form
Competent cellcapable of taking up DNA
May be important route of genetic exchange in nature
DNA bindingprotein
nuclease – nicks and degrades onestrand
competence-specificprotein
Streptococcus pneumoniae
Bacteria and transformationBacteria and
transformation Not all bacteria
can be transformed in nature
Streptococcus pneumonia, Haemophilus influenza, and Neisseria gonorrhea
Not all bacteria can be transformed in nature
Streptococcus pneumonia, Haemophilus influenza, and Neisseria gonorrhea
Transformationhttp://www.dnalc.org/ddnalc/resources/transformation2.html
Transformationhttp://www.dnalc.org/ddnalc/resources/transformation2.html
Uptake of DNA can only occur at a certain cell density
Cells need to be in the log phase of growth
A competence factor is required for the uptake of DNA from the environment
Uptake of DNA can only occur at a certain cell density
Cells need to be in the log phase of growth
A competence factor is required for the uptake of DNA from the environment
Genetic recombination and transformation in the
laboratory
Genetic recombination and transformation in the
laboratory Plasmids are designed to
contain genes of interest Transformation done in
laboratory with species that are not normally competent (E. coli)
Variety of techniques used to make cells temporarily competent calcium chloride treatment
makes cells more permeable to DNA
Plasmids are designed to contain genes of interest
Transformation done in laboratory with species that are not normally competent (E. coli)
Variety of techniques used to make cells temporarily competent calcium chloride treatment
makes cells more permeable to DNA
Cloning vectorsCloning vectors
pGlo and transformationpGlo and transformation
Lab protocolLab protocol Obtain two tubes containing CaCl2 Label One tube +DNA, Label the other tube –
DNA/Group These tubes have been on ice for one hour+ Add your bacteria cells and incubate for thirty Pick bacterial colonies or cells and add them to both the
+ and – tubes Vortex the tube and replace on ice To the + tube add plasmid DNA 10 ul of either green or blue 5ul of blue and green Do not add plasmid to the – DNA tube Check tips to make sure that you added the plasmid to your cells Mix by pulsing in the microcentrifuge
Obtain two tubes containing CaCl2 Label One tube +DNA, Label the other tube –
DNA/Group These tubes have been on ice for one hour+ Add your bacteria cells and incubate for thirty Pick bacterial colonies or cells and add them to both the
+ and – tubes Vortex the tube and replace on ice To the + tube add plasmid DNA 10 ul of either green or blue 5ul of blue and green Do not add plasmid to the – DNA tube Check tips to make sure that you added the plasmid to your cells Mix by pulsing in the microcentrifuge
Heat shockHeat shock
Incubate for thirty minutes on iceKeep your tubes in ice in a cup and
go to water bathHeat shock at 42oC for 90 secondsRemove tubes from bath and
immediately place back on ice for 2 minutes
Incubate for thirty minutes on iceKeep your tubes in ice in a cup and
go to water bathHeat shock at 42oC for 90 secondsRemove tubes from bath and
immediately place back on ice for 2 minutes
RecoveryRecovery
Add 250 ul of the Luria Broth to the transformation tubes. The Luria Broth is rpewarmed. It should incubate for at least fifteen minutes at 37oC. Place your tubes in the incubator.
Add 250 ul of the Luria Broth to the transformation tubes. The Luria Broth is rpewarmed. It should incubate for at least fifteen minutes at 37oC. Place your tubes in the incubator.
ProtocolProtocol
Preparation of platesTwo plates should be labeled + DNA+DNA LB
+DNA-LB’AMPTwo plates should be labeled – DNA
as aboveAdd 250 ul of transforming solution
and Luria to each plate
Preparation of platesTwo plates should be labeled + DNA+DNA LB
+DNA-LB’AMPTwo plates should be labeled – DNA
as aboveAdd 250 ul of transforming solution
and Luria to each plate
Spread PlatesSpread Plates
Make a spread plate by spreading the 250 ul of sample first horizontally, then vertically , and finally diagonally.
Stack plates and tape. Let plates sit bottom side down until fluid is absorbed into the agar.
Make a spread plate by spreading the 250 ul of sample first horizontally, then vertically , and finally diagonally.
Stack plates and tape. Let plates sit bottom side down until fluid is absorbed into the agar.
Incubate overnightIncubate overnight
Incubate overnight at 37oC.Check for growthCheck selection plates for
transformants Use the long range uv light to
check for fluorescence.
Incubate overnight at 37oC.Check for growthCheck selection plates for
transformants Use the long range uv light to
check for fluorescence.