Green fluorescence protein application

1. GFP: Application in Bacterial dynamics & disease diag
PRESENTED BY: Dr. GARIMA SHRINET
PhD scholar, Department of Veterinary Microbiology, LUVAS, Hisar

2. Introduction
 GFP was discovered as companion protein to aequorin , Aequorea
victoria
( Shimomura et al., 1962)
 Chromophore is 4-(p- hydroxybenzylidene ) imidazolinone
 Crucial breakthrough came by cloning of GFP gene
( Prasher et al., 1992)

3. Noble prize in chemistry,2008
OSAMU
SHIMOMURA
MARINE BIOLOGICAL
LABORATORY,
WOODS HOLE ,USA,
BOSTON UNIVERSITY
MARTIN CHALFIE
COLUMBIA UNIVERSITY
,NY,USA
ROGER Y. TSIEN
UNIVERSITY OF CALIFORNIA.
SAN DIEGO, CALIFORNIA ,USA

4. Mechanism of fluorescence in jelly fish
Ca3- APO-AEQUORIN- COELENTERAMIDE
AEQUORIN +
COELENTERAZINE
Blue
light
Green
light
+3Ca 2+ & coelenterazine oxd.
+ GFP
(Marc Zimmer ,2001)

5. Structure of GFP
( Roger Y. Tsien,1998)

6. How fluorophore become active

7. GFP derivatives
 wild type GFP excitation at 395nm & emission at 508 nm
 S65 T ( Y. Tsien ,1995)
 RED Shift
(Roger heim ,1996)

8. Excitation & emission wavelength
FP Excitation (nm) Emission (nm)
EGFP 488 507
BFP 308 440-447
CFP 458 480
ds RED 557 585
mcherry 587 610
YFP 525 538
Fluorescence excitation & emission spectra of native GFP from Aequorea victoria (Tsien et al.,
1998).

9. Classification of GFP
 GFP divided into 7 classes ,component of Chromophore
 Class 1 – wild type
 Class 2 – phenolate anion
 Class 3 – neutral phenol
 Class 4 – phenolate anion with stacked Π electron system
 Class 5 – indole
 Class 6 – imidazole
 Class 7 - phenyl
(Tsien et al., 1998).

10. Characteristics of GFP
 Expressed efficiently
 No phototoxicity
 Sufficient photostability
 Minimal overlap in excitation & emission
 Does not require cofactor or substrate
 GFP is resistant to heat, alkaline pH,detergents, photobleaching.
(Orm¨o et al. , 1998 as per PDB)

11. Fusion tags
Fusion between cloned gene &GFP
Chimera be expressed in cell or organism
Visualize dynamic cellular events& monitor protein localization
Chimeras created fusing protein of interest to C or N termini
GFP created with new C& N termini in 10 different positions
E142, Y143,Y145, H148, D155, H169, E172, D173, A227, &I229

12. Transformation pGLO
Ara C - turns on GFP in presence of arabinose
GFP - make organism glow
Bla - break ampicillin
Arabinose in media activate GFP
Ampicillin kills any bacteria that does not have pGLO

13. GFP as a selectable marker for antimicrobial
peptide clone
 11-residue antimicrobial peptide from bovine lactoferrin (BL-11) and the 12-
residue hypotensive peptide from αs1-casein (C-12).
 cloned in Streptococcus thermophilus to develop strains that enhance the
functionality and nutritional value of dairy food products.
 Nucleic acid sequences encoding the peptides were generated by overlapping
PCR.
 S. thermophilus transformants were successfully identified using GFP as a
selectable marker
J.R Renye et al., 2007

14. GFP IN DIVA
 The use of effective vaccines and the corresponding diagnostic tests that allow
differentiating infected from vaccinated animals are essential tools to control the
disease
 a prototype of Brucella abortus S19 vaccine expressing green fluorescent protein (S19-
GFP) was constructed
 The S19-GFP was readily identified under ultraviolet light by macroscopic and
microscopic examination and maintained all the biochemical characteristics of the
parental S19 vaccine.
 S19-GFP replicated ex vivo and in vivo, and protected mice against challenge with
virulent B. abortus to the same extent as the isogenic S19
 Both vaccines raised antibodies against lipopolysaccharide molecule to similar levels
(Carlos chaon et al.,2010)

15. GFP reporter of gene transcription
 Measure gene expression in real time, at single cell level
 No exogenous substrate is required
 Measure fluorescent level by FACS
 Reveal heterogeneity in bacterial population

16. GFP fusion for Protein localization &
Dynamics Numerous cytoplasmic protein fusions to GFP , used in vivo
 Successful transport of GFP to periplasm , using Tat pathway( Twin Arginine
Pathway) (Thomas et al., 2001)
 FCS & FRET characterize state of signal transduction in real time

17. Role of Min protein in E.coli cell division
 GFP – Min D fusion engage in oscillatory behaviour
(Raskin & de Boer et al.,1999)
 Protein relocates every 10-30 secs, fluorescence appears ill- defined
 MinE oscillation to remove MinC & D , the division inhibitor, from
cell division (Fu et al., 2001)

18. Signal transduction protein in Caulobacter
crescentus
 CckA localizes to pole in pre divisional cell ,disperses before cell
division
(Jacob et al, 1999)
 GFP , YFP,CFP helped in localization of PleC , DivJ
 Cell processes in bacteria regulated by differential localization of
components of signal transduction network

19. Chromosome and plasmid segregation
 GFP-fused partitioning protein binding ori , & GFP – lacI repressor
hybrid
 One sister ori stays near cell pole , while other to new pole in
daughter cell
( G.S Gordon, D. Sitnikov, 1997)
 Model of segregation of high copy number suggest that they are free
to diffuse in cytoplasm
 Ori of both E.coli &B .subtilis associate at or near the cell pole early
in cell cycle ( C.D Webb, A. Teleman et al.,1997)

20. Budding of Yeast
 Individual chromosomal loci detected expression of protein fusion ,
GFP & Lac repressor
 Spindle microtubules detected , GFP and Tub1, the major α tubulin
 Spindle elongation &chromosome separation exhibited biphasic
kinetics
 Budding yeast did not exhibit metaphase chromosome , did show
 Anaphase (Araon F.Straight,John W. Sedat,1997)
Spindle pole bodies

21. Bacterial Sporulation
 Formation of forespore & mother cell
 GFP fusion with CotA & DacF
 Spo1V A localization, Time - lapse photomicroscopy
 Spo1VA - mother cell membrane surrounding forespore
 GFP – SpoIIE , to explain σF selectively activated in
developing spore
(O.King, Stragier, R. losick ,1999)
Septation, dephosphorylation, & activation of σF
during sporulation in Bacillus subtilis

22. Spatial pattern of gene expression in
Bacterial Biofilm
 Biofilm leads heterogeneity ,result into spatial & temporal
pattern
 Multiple spp. , defined spatial distribution
( Aspiras et al., 2000 )
 Growth dependent promoter (rrnBP1) fused with destabilized
GFP variant (Sternberg et al., 1999)
 Quorum sensing GFPmut3 gene, AHL mediated
Spatial organization in mixed-species biofilms.

23. Identification of Bacterial gene expressed in
Host
 Invasion & Intracellular survival
 Salmonella typhimurium random promoter with promoterless GFP
gene where used to infect Macrophages , FACS
 Fluorescence cells were lysed,FACS analysis in absence of host cell
 Confirmed the intracellular dependence of gene induction
( Valdivia &Falkow, 1997)
Image showing Salmonella typhimurium injection

24. Mycobacterium tuberculosis reporter
strain
 Mycobacterium tuberculosis senses chloride & pH
(Tan, S., Sukumar, N., Abramovitch & Russell, D.G.
2013)
 rv2390c: [Cl-] and low pH
 rv2390c’::GFP a Sensor for phagosome maturation
 hspX: Hypoxia and NO
HIV /TB coinfection

25. The image shows Mycobacterium tuberculosis transformed to express mCherry (red)
constitutively, and GFP (green) under regulation of a pH-sensitive promoter

26. GFP to investigate Ag nanoparticle
antibacterial activity
 Recombinant E. coli bacteria expressing GFP, model system
 To investigate the antimicrobial activities of Ag NPs
 Rapid detection of time-dependent changes in bacterial growth
fluorescence characteristics in the presence of Ag NPs
(P.Gopinath, A.Paul, A. Ramesh,2006,IIT Bombay)

27. CAMELEONS
 Dynamically responsive biological indicators based on Ca 2+
(Romoser et al.,1997)
 Linked BFP &GFP with 26 residue spacer containing CaM-
domain from avian smooth muscle myosin light chain kinase
 Addition of Ca –CaM disrupted FRET ,decrease the 505 nm
emission by 65% & ratio of 505 to 440 nm by 6 fold
 M13 & CaM
( Miyawaki et al.,1997)

28. Advantage of GFP- Ca2+ indicators
 Applicable to all organisms
 Targeted to specific tissue, cells, organelle
 Do not blur spatial gradient
 Good optical properties
Disadvantage
 Gene transfection is required
 Binding kinetics is slower
 CaM &M13 may have additional biological activitiy

29. FRET biosensors
Biosensor construct
Cameleons
GFP type
CFP, YFP
Fluorescence
measurement
FRET
Detected
Cytoplasmic and
organelle Ca2+
indicators
FIP–CBSM BFP, GFP FRET
Intracellular Ca2+
indicator
GFP–Bax,
BFP–Bcl-2
BFP, GFP FRET
Apoptosis
detection
(protein–protein
interaction)
(Steve R. Kain ,1999)

30. Study of protein interaction

31. GFP Vs IFM
 Requirement for antibody
 Require cell fixation
 Technically demanding
 GFP hightens sensitivity, good for Two photon Excitation
 Autocyclization, no invasive sample preparation
 Protein localization monitored in real time,viability assay

32. GFP Caveats
 Detection at low level of expression is difficult
 Dependent on oxygen & pH
 Overexpression lead to mislocalization
 GFP mutants still express lag
 Temperature sensitive GFP folding

33. GFP use outside the laboratory….
 Used in monitoring meat fermenting
lactobacilli in sausages
 Tracking spread of bacteria that consume fuel
in soil
 In vivo cancer imaging, metastatic movement
 ANDi , Noel pig, GFP Bunny
 BRAINBOW

34. CONCLUSION
 GFP - wonderful molecule
 Discovered from depths of ocean, revolutionary light in
molecular biology
 Innovative application, provide window into complexity
of bacterial cell
 Help in understanding bacterial pathogenesis
 Providing new insights in understanding internal
workings of bacteria

35. THANK YOU