1. Green Fluorescent Protein Molecular Genetics

2. Green Fluorescent Protein  Green Fluorescent Protein (GFP) has existed for more than one hundred and sixty million years in one species of jellyfish, Aequorea victoria

3. Fluorescence  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.

4. Fluorophore  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.

5. Fluorophore  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.

6. Absorption spectrum of gfp

7. Excitation and Emission

8. Amino acid Sequence gfp  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//

9. Blue 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

10. Blue 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.

11. Chameleons

12. Different mutations causes different colors

13. Fluorescence in Nature

14. Fluorescent Molecules used in research

17. Fluorescence in Research

18. DNA Transformation  Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form  Competent cell  capable 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 cell  capable of taking up DNA  May be important route of genetic exchange in nature

20. DNA binding protein nuclease – nicks and degrades one strand competence-specific protein Streptococcus pneumoniae

21. Bacteria 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

22. Transformation http://www.dnalc.org/ddnalc/resources/transformation2.html http://www.dnalc.org/ddnalc/resources/transformation2.html Transformation http://www.dnalc.org/ddnalc/resources/transformation2.html http://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

23. 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

24. Cloning vectors

25. pGlo and transformation

26. Lab 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

27. Heat shock  Incubate for thirty minutes on ice  Keep your tubes in ice in a cup and go to water bath  Heat shock at 42 o C for 90 seconds  Remove tubes from bath and immediately place back on ice for 2 minutes  Incubate for thirty minutes on ice  Keep your tubes in ice in a cup and go to water bath  Heat shock at 42 o C for 90 seconds  Remove tubes from bath and immediately place back on ice for 2 minutes

28. Recovery  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.

29. Protocol  Preparation of plates  Two plates should be labeled + DNA  +DNA LB +DNA-LB’AMP  Two plates should be labeled – DNA as above  Add 250 ul of transforming solution and Luria to each plate  Preparation of plates  Two plates should be labeled + DNA  +DNA LB +DNA-LB’AMP  Two plates should be labeled – DNA as above  Add 250 ul of transforming solution and Luria to each plate

30. Spread 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.

31. Incubate overnight  Incubate overnight at 37oC.  Check for growth  Check selection plates for transformants  Use the long range uv light to check for fluorescence.  Incubate overnight at 37oC.  Check for growth  Check selection plates for transformants  Use the long range uv light to check for fluorescence.