Transcript Green Fluorescent Protein

Green Fluorescent Protein
Molecular Genetics
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
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.
 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 alphahelix 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.
Fluorophore
Fluorophore
 The fluorophore itself is a p-
hydroxybenzylideneimidazolidone. 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 SerTyr-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
Excitation and Emission
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//
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
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.
Chameleons
Different mutations causes
different colors
Fluorescence in Nature
Fluorescent Molecules used
in research
Fluorescence in Research
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
Streptococcus pneumoniae
DNA binding
protein
competence-specific
protein
nuclease – nicks and degrades one
strand
Bacteria and transformation
 Not all bacteria can
be transformed in
nature
 Streptococcus
pneumonia,
Haemophilus
influenza, and
Neisseria
gonorrhea
Transformation
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
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
Cloning vectors
pGlo and transformation
Lab protocol
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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 shock
 Incubate for thirty minutes on ice
 Keep your tubes in ice in a cup and go
to water bath
 Heat shock at 42oC for 90 seconds
 Remove tubes from bath and
immediately place back on ice for 2
minutes
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.
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
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.
Incubate overnight
 Incubate overnight at 37oC.
 Check for growth
 Check selection plates for
transformants
 Use the long range uv light to check
for fluorescence.