Green fluorescent protein of the jellyfish Aqueorea victoria is a 238-amino-acid, 28-kDa protein that absorbs light with an excitation maximum of 395 nm and fluoresces with an emission maximum of 509 nm (Shimomura et al, 1962). GFP owes its unique spectral properties to its chromophore (Ormö et al,1996) that consists of a Ser65, Tyr66, and Gly67 tripeptide (Cody et al, 1993). Autocatalytic cyclization of this tripeptide, induced by oxidation of Tyr66, is a necessary posttranslational step for proper fluorescence (strong reducing agents reversibly convert GFP into a nonfluorescent form) (Heim et al, 1994). This can occur in the absence of any cofactors, making GFP an extremely useful tool for a wide range of applications in a variety of heterologous systems (Ausubel, et al, 1994). GFP activity can be assayed both qualitatively and quantitatively using a variety of techniques, including simple plate counting, fluorescence and confocal microscopy, flow cytometry, and fluorometry. Transcriptional and translational fusions of GFP to a gene or protein of interest can be used as gene expression reporters and subcellular localization tags. GFP is a small protein (28 kDa) compared to other reporters (e.g., β-galactosidase is 465 kDa) and GFP fusions often retain the native protein function (Chalfie, et al,1998). This makes GFP useful as a generic tag for studying protein synthesis, translocation, and other protein-protein interactions. GFP is also widely used as a reporter in many genetic techniques, including transposon mutagenesis, promoter/enhancer traps, and one-component hybrid systems. GFP can be visualized using microscopy in both live and fixed cells, making it an excellent tool for studying dynamic changes in living cells.
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Recombinant DNA is simply any DNA molecule composed of sequences derived from different sources.the key to cloning a DNA fragment of interest is to link it to a vector (plasmid vectors in E.coli) DNA molecule, which can replicate within a host cell. After a single recombinant DNA molecule , composed of a vector plus an inserted DNA fragment , is introduced into a host cell. The inserted DNA is replicated along with the vector , generating a large number of identical DNA molecules.
Vector + DNA fragment
Replication of recombinant DNA within host cell
Isolation , sequences and manipulation of purified DNA fragment
A major objective of DNA cloning is to obtain discrete , small regions of organisms DNA that constitute specific genes. In addition, only small DNA molecules can be cloned in any of the available vectors. Two types of enzymes --- restriction enzyme and DNA ligases facilitate production of such recombinant DNA molecules .restriction enzyme from E.coli makes staggered cuts at the specific 6-pb inverted repeat sequence shown , yielding fragment with single - stranded, complementary 'stick' ends (figure 1)
Figures 1 to be added
Then, vector DNA cut with EcoRI is mixed with a sample containing restriction fragments produced by cleaving genomic DNA with several different restriction enzymes. The short base sequences composing the sticky ends of each fragment type are shown. The sticky end on the vector DNA (a') base -pairs only with the complementary sticky ends on the EcoRI fragment (a) in the genomic sample. The adjacent 3'-hydroxyl and 5'-phosphate groups on the base -pairs fragments then are covalently joined(ligated) by T4 DNA ligase( figure 2)
Figure 2 to be added
Plasmid vectors contain a selectable gene such as ampr , which encodes the enzyme β- lactamase and confers resistance to a ampicillin. Exogenous DNA can be inserted into the bracketed region without disturbing the ability of the plasmid to replicate or express the amp gene. Plasmid vectors also contain a replication origin (ORI) sequence where DNA replication is initiated by host cell enzymes. Inclusion of a synthetic polylinker containing the recognition sequences of several different restriction enzymes increase the versatility of plasmid vector. The vector is designed so that each site in the polylinker is unique on the plasmid ( figure 3).
Figure 3 to be added
Material and methods:
The procedure was provided with below materials
2µl EcoRI/HindIII cut and cleaned PUC19 vector
5µl EcoRI/HindIII cut and cleaned GFP insert
2µl 10xT4 ligase buffer
2µl T4 ligase (0.5 U ml-1)
Sterile H2O to make up to 20µl volume (9
Always on Time
Marked to Standard
µl is used in this procedure)
--The ligation reaction
Is performed from mixing of the above materials and into concentration of 20µl of the ligation reaction then this ligation reaction mixture is incubated at room temperature for 30 minutes .After that ,
--Transformation of ligation into cloning host :
Is done via defrosting 100µl of competent BL21 E. coli on ice, these then followed by addition of 10µl of the ligation reaction into competent cells and then incubated on ice for 30mins , the transformation mixture takes out of ice and heat shock at 42 °C for 75 seconds. Following the heat shocking return immediately into ice for a minimum of 2 mins ,plate out cells on a selective media plates(1.5 % LB agar ,4oµg ml-1 x-gal,0.1mM IPTG ,50µg ml-1 ampicillin).then allow transformation to dry out onto plates and incubate agar up at 37°Cfor 12-18 hours.
--Picking of colonies for protein expression
Set up 2x5ml LB +50µg ml-1 ampicillin in 30ml sterile tubes, then pick up 1xBlue individual colony and 1x white individual colony and inoculate separate tubes. Then incubate in shaking incubator overnight at 37 °C , 220rpm .
--Subculture and growth of recombinant E.coli for protein production
Firstly ,prewarm 2x60ml sterile LB,in 250ml conical flask ,(1 per inoculum )at 37 c ,then add ampicillin to a final concentration of 50µg ml -1 ampicillin aseptically ,then remove 1ml of media and place in cuvette to act as blank ,followed by addition of 600µl overnight to calture of each individual colony to separate flask (1:100 inoculum) ,next return flask to shaking incubator and incubate at 37 c ,200 rpm ,then blank spectrophotometer at 600nm against media ,then at 45 min interval remove sample aseptically from flask for each flask remove 1x 1ml to add a fresh clean cuvette (take to next stage 8) and 1x1ml into clean eppendrof take to stage 9) .Measure the OD600nm of culture in cuvette and record result for growth curve(once culture reached have reached a OD 600nm of 0.5,add IPTG to final concentration 1Mm stock solution. Spin down samples in eppendrof tube at max speed in microcentrifuge for 5 mins , ensure centrifuge is balanced before spinning (remove supernatant and retain pellet and resuspend pellet in 200µl cell lysis buffer(10mMl Tris PH8.0,300Mm Nacl ,10mg ml-1 Lysozyme.Freeze resuspended cells at -20 c following day.finally continue sampling until OD600nm has no longer risen for two successive samples.