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Gfp application in bacterial dynamics and disease diagnosis
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)
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
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