Over current years there has been an explosion of interest in the use of micro-organisms and other cell-based systems for the production of biological products and small molecules. The list of recombinant proteins now routinely used in medicine is huge and apparently ever growing. Green Fluorescent protein (GFP) is a protein which was first isolated from a jellyfish named Aqueorea Victoria however it is present in other marine animals too. The GFP is composed of 238 amino acid, 28-kDa protein exhibits green fluorescence light when it is exposed to light in blue within ultra violet range with an excitation of 395 nm and fluorescence with an emission of 509 nm (Shimomura et al, 1962). Green Fluorescent protein has very distinctive spectral characteristics to its chromophore (Ormö et al, 1996) and comprises of Ser65, Tyr66, and Gly67 tri-peptide (Cody et al, 1993). Oxidation of Tyr66 leads to an autocatalytic cyclization of this tri-peptide, this step is essential for the post translational step for the proper production of fluorescence (powerful reducing agents reversibly change GFP into a non fluorescent state) (Heim et al, 1994). This process does not require any additional co-factor, which makes GFP a very helpful agent for extensive purposes in different heterologous systems (Ausubel, et al, 1994). Performance of GFP can be observed by a variety of methods for knowing both the qualitative and quantitative properties. Such techniques include: simple plate counting, fluorometry, flow cytometry, fluorescence and confocal microscopy. Fusing GFP by transcriptional and translational means to a gene or a protein is used in expressing genes reporters and sub cellular localization tags. When we compare GFP to other reporters like β-galactosidase which is a 465 kDa it is found that Green fluorescence protein is comparatively smaller than them and their fusions mostly maintain the main function of protein (Chalfie, et al, 1998). This contributes in making GFP a helpful tool for understanding protein synthesis in a better way and other process like translocation and many other protein-protein interactions. GFP is commonly used in many genetic techniques as a reporter, also includes transposon mutagenesis, promoter/enhancer traps, and one-component hybrid systems. Visualization of Green Fluorescent protein (GFP) in both live and fixed cells can be done by microscopy, which makes it the best means of knowing about the dynamic changes that occur in the living cells.
Recombinant DNA can be defined in simple words as any DNA molecule that comprises of the series obtained from diverse sources. The factors that adds up to a success in cloning a DNA fragment includes linking it to a vector (plasmid vectors in E.coli) DNA molecule, which has ability to replicate in a host cell. As soon as a single recombinant DNA molecule, that consists of a vector and an inserted fragment of DNA, is included in a host cell. This inserted DNA starts replicating along with the vector, producing similar DNA molecules in greater numbers. The following describes the whole process:
Vector + DNA fragment
Recombinant DNA
Replication of recombinant DNA within host cell
Isolation, sequences and manipulation of purified DNA fragment
The main aim of DNA cloning is to get discrete, small regions of species DNA that contain specific genes (Lodish, et al, 1986). The DNA ligases and restriction enzymes together facilitate formation of this recombinant DNA. Restriction enzymes from E.coli create cuts at the 6-pb inverted sequence as shown below; this yields a single fragment which is stranded with complementary 'stick' ends (figure 1)
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Figure 1 (Lodish , et al, 1986)
Later, the vector DNA that is cut with EcoRI is merged with the sample of restriction fragments yielded by breaking of DNA with various restriction enzymes. Then, the small base sequences combining the sticky ends from each fragment are shown. It is observed that only the sticky end of the vector DNA (a') pairs with the complementary sticky ends of the EcoRI fragment (a). Whereas 3'-hydroxyl and 5'-phosphate groups on the base pairs with fragments and are covalently bonded by DNA ligase (figure 2).
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Figure 2 ( Lodish, et al, 1986)
Plasmid vectors are genes like ampr which encodes β- lactamase enzyme and provides resistance to ampicillin. DNA from exogenous sources can also be inserted. In addition Plasmid vector also has a replication origin (ORI) sequence in which replication of DNA started by the host enzymes. Including an artificial polylinker that possess the recognition sequences elevate versatility of these plasmid vectors. Each site in the polylinker is different due to the exclusive design on the vector (figure 3).
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Figure 3 (Lodish, et al, 1986)
This experiment aimed to illustrate cloning by injecting a gene for the Green Fluorescent protein into a bacterial plasmid vector pUC18/19 so as to produce a green color light when observed under fluorescence microscope.
Materials and Methods
Materials
Following materials were used for the procedure:
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
µl required in this procedure)
Methods
*Ligation Reaction
It starts with mixing the above mentioned materials into 20µl concentrated of the ligation reaction, after that this mixture is left at room temperature for about 30 minutes.
*Transformation of Ligation into Cloning Host
It is a simple process and is conducted by defrosting 100µl of BL21 E.coli on frost, and then adds 10µl of the ligation reaction to competent cells. They are then inoculated for almost 30 minutes over ice. The transformed mixture is taken out of the ice and heated at 42 °C for duration of 75 seconds. Now, allow the transformed sample to dry out and incubate on agar for 12-18 hours at 37°C.
*Selection of Colonies for Protein Expression
Prepare 2x5ml LB +50µg ml-1 ampicillin in 30ml sterile tubes, then select 1xBlue individual colony and 1x white individual colony into separate tubes. Now, incubate in a shaking incubator throughout the night at 37 °C, 220rpm.
*Subculture and Growth of Recombinant E.coli for Protein Formation
First of all, warm 2x60ml sterile LB, in 250ml conical flask, ,(1 per inoculums )at 37 c , after that include ampicillin to a last concentration of 50µg ml -1 ampicillin avoiding contamination with bacteria, then eradicate 1 ml of media and put in a cuvette , now keep adding throughout the night 600µl to each culture, put the flask back to the shaking incubator, after that place blank spectrophotometer against media at 600nm , remove this sample after 45 minutes , in every flask remove 1x 1ml and keep adding pure cuvette ( take to next stage 8). Calculate the OD600nm of the culture and note the outcome for the growth curve (one culture have arrived at an OD 600 nm of 0.5; include IPTG to the final concentration. Rotate the samples in a centrifuge for 5 minutes at its maximum speed. Make sure the centrifuge is in equilibrium prior to spinning ( eliminate retained pellet and re suspend pellet in 200µl cell lyses buffer (10mMl Tris PH8.0,300Mm Nacl ,10mg ml-1 Lysozyme. Ice up resuspended cells on the same day at -20C. Keep sampling till OD600nm stops rising for two consecutive samples.
Result and discussion :
The practical class was carried out on BL21 strain of E.coli in order to express the protein of interest, GFP was insufficient due to some technical problem during the procedures.
1-ligation phase:
In normal experiment the vector should be ligated to the DNA fragment to be cloned, by using DNA ligase which is obtained from T4 infected E.coli. Ligation is regularly carried out at lower temperatures to support annealing over an extended a period of time for several hours to allow the DNA ligase to work (Reed, et al, 1998).
In this experiment, the ligation phase has done wrongly due to several reasons such as ;
T4 DNA ligase was not enough stored at -20 C' or stored below.The T4 DNA ligase enzyme is an extremely temperature sensitive.
T4 DNA ligase could be inactivated.
ATP energy in the buffer reaction might be degraded.
The insert and plasmid vector have incompatible ends.
2- Transformation:
It is described by when a recombinant plasmid vector has been created , it should be introduced into an appropriate host cell. However, because the first step goes wrong ( ligation phase) the transformation as well. Because of some reasons;
Time of heat shocking could be not enough to transformation of E.coli .
Water bath temperature may be less than 42C ( this temperature is suitable for transformation ) or a higher , which decrease in transformation efficiency ( Smith, et al, 1992).
Transformation efficiency calculated by :
Number of colonies on plate divided ng of DNA multiply 1000ng/µg = (transformation/ µg)
1 µg = 0.0001 ng.
The cells were handled improperly between heat shock and ice or the cells was insufficient .
May be there was excess of DNA concentration.
3- Growth curve:
Normally growth curve show S- shaped when plotted in log linear format ( figure 4) which divided into four components
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Figure 4 ( Reed, et al, 1986)
Lag phase ; the initial period when no increase in cell number is seen.
Log phase; when cells are growing at the maximum rate.
Stationary phase; growth decreases as a nutrient are depleted and waste products accumulate.
Death phase; this is the result of prolonged starvation and toxicity.
In this experiment , the growth curve showed a significant difference compared to the typical one . It means there was an error could be from the IPTG induction. The inducer may be kept in warm condition or could be an old, which given poor induction. Further, stationary phase at which bacteria waste product in medium becomes high and hold back bacteria to stay alive, it looks in figure 5 completely abnormal stationary level i.e. bacteria are failing rapidly and resulting in death phase later.
Figure 5
4- Protein expression:
Protein expression could be affected by a number of reasons including; vectors, host strain and growth condition. Some codons are rarely used by bacteria in DNA cloning , during the insertion of this codon could be replaced the normal one and resulting of inability to transfer protein (Lodish, et al, 1986). Furthermore, unsuccessful ligation to Lacz gene which could not disrupt the function of β- galactosides and resulting in the blue colonies (Joung, et al, 2000). In term of selection of colonies (blue and white ), it has difficult to pick a white colony up because it was not obvious. In addition, frame shifting of DNA nucleotide, deletion mis-corporation such as lysin for origenin can have a significant impact for the expression of protein levels. In addition of that, protein expression could be affected by DNA impurities ( contaminations).
Conclusion:
The experiment was aimed to produce a protein which given a green color light when observed under fluorescence microscope. However, due to some technical errors during the procedures from ligation to expression of protein, the process went unfortunate outcome. Escherichia coli is one of the most commonly used hosts for the creation of heterologous proteins and its heredity are far better characterized than those of any other microorganism .
