Recombinant DNA technology, popularly called genetic engineering, began around 1973 in the effort of Stanley Cohen and Herbert Boyer Cohen et al. 1973. In their study, they made use of Escherechia coli plasmid pSC101 to clone DNA from Salmonella typhimurium streptomycin resistance plasmid RSF1010. A few years later, the technique has advanced through Genentech, a biotechnology company, when they started to produce a human recombinant protein. Their first successful work was the gene expression encoding human somatostatin in E. coli (Itakura et al. 1977). The bioactive substance produced was akin to that of somatostatin extracted from the brains of 500.000 sheep. Furthermore, Genentech followed up this success in 1982 when they produced recombinant insulin called humulin which was also the first recombinant biotechnology drug to be accepted by the Food and Drug Administration for market. In the present time, the demand for recombinant protein production has become so great that it can reach about $50 billion yearly (Schmidt 2004).
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Along with the birth of recombinant DNA technology is the prominence of the two bacterial hosts for gene expression, Escherichia coli and Bacillus spp. These hosts were dominantly used in different genetic engineering studies even until today. Nonetheless, scientists have realized that different proteins may require different host physiology and biochemistry for optimal production leading to the search for new hosts, both prokaryotic and eukaryotic. Advancing of the recombinant DNA technology has opened the use of novel organisms which lead to many different expression systems in many different hosts. To name are systems for use in gram-positive bacteria Streptomyces (Binnie et al. 1997), insect and animal cell cultures (Wurm 2004; Kost et al. 2005), filamentous fungi (Nevalainen et al. 2005), and in yeasts (Gellissen et al. 2005).
Among the many expression systems in use today, still, bacterial expression systems and the preferred choice for producing many prokaryotic and eukaryotic proteins. Others may also give good results but bacteria are most cost-effective, has well-characterized genetics, and there are a number of different expression systems for bacteria. Among all bacterial hosts available for recombinant expression, the use of E. coli still remains the most exemplary. This is because intense research has been made on the genetics of this host for over many decades. Further is the availability of broad biotechnology tools for the genetic engineering of this organism (Yin et al. 2007).
As a most valued host, E. coli's favoured characteristics include its (a) fast rate of growing; (b) cheap culture media cost; (c) capacity to continuously ferment; and (d) high expression levels are achievable for this host (Yin et al. 2007). As such, 80% of all proteins for solving three-dimensional structures were prepared in E. coli (Sørensen and Mortensen 2005) which were submitted to the protein data bank (PDB) in 2003. Also, between 2003 and 2006, 9 out of 31 therapeutic proteins that were approved were produced in E. coli (Walsh 2006). Among these are two important growth factors, namely insulin and interferon (Schmidt 2004).
Separately, another effort was made for Green Fluorescent Protein (GFP) in 1962. This substance, isolated from the Aequorea aequorea (Shimomura et. al., 1962) was found as a protein accompanying aequorin, a well-known chemiluminescent protein of the same jellyfish species. In the study of Tsien in 1998, the emission spectrum of a live tissue of A. aequorea peaks at 508nm which looks green. However, that of a pure aequorin peaked at 470 nm, in the blue range. The result of this study has made the group of Shimomura's to discover GFP suggesting a mechanism for exciting the protein which they called radiation-less energy transfer. Its structure consists of an 11-stranded Î²-barrel containing the chromophore made up of a single Î± helix shown in Figure1.
Figure 1. The structure of GFP.
The use of GFP as a molecular biology tool was not realised until Prasher reported the cloning and sequence of GFP (Prasher, et al. 1992) in 1992. Two years after, GFP has been used as a reporter protein (Chalfie et al. 1994) by emitting green light at Î»em = 508 nm upon excitation with either near ultraviolet light (around 395 nm) or blue light (around 470 nm) (Ito et al. 1999). Later, several mutations aiming to (a) improve the emission; (b) focus to a single wavelength (Heim, et al. 1995); and (c) change the colour of the emitted light itself have been made.
Recombinant DNA molecules usually contain a DNA fragment inserted into a bacterial vector. With the use of Polymerase Chain Reaction (PCR), a speciï¬c gene or DNA region of interest can be isolated and ampliï¬ed by DNA polymerase extracted from a heat-tolerant bacteria. PCR "ï¬nds" target DNA (DNA region of interest) by complementary binding speciï¬c short primers to the ends of that sequence. The long chromosome-size DNA molecules of genomic DNA must be cut into fragments of a much smaller size before they can be inserted into a vector. With the use of bacterial restriction enzymes, cutting is done. These cut at speciï¬c DNA sequences, termed as restriction sites. This property is the reason that makes restriction enzymes appropriate for manipulating DNA, examples of which are endonucleases cleaving a phosphodiester bond. To mention is EcoRI which recognizes the following sequence having six nucleotide pairs: 5'-GAATTC-3' and 3'-CTTAAG-5'.
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The EcoRI is an enzyme that cuts only between the G and the A nucleotides on each strand of the palindrome. The recombinant DNA molecules are transferred into bacterial cells, with each cell taking up only one recombinant molecule. This is ampliï¬ed along with the vector during bacterial cell division. The result then is a clone of identical cells, each containing the recombinant DNA molecule; thus, this amplification technique is called DNA cloning.
Bacterial plasmids are small circular DNA molecules which replicate their DNA separated from that of the bacterial chromosome. These are used routinely as vectors which carry (a) a gene for drug resistance; and (b) a gene to distinguish plasmids with and without DNA inserts. These genes which are resistant to the drug (antibiotic) provide a useful way to choose for bacterial cells transformed by plasmids. Live cells after exposure to the drug are expected to carry the plasmid vectors. However, not all the plasmids in these transformed cells will contain DNA inserts; thus, it is ideal to be able to recognise colonies with plasmids containing DNA inserts. DNA inserts disrupt a lacZ gene in the plasmid encoding -galactosidase, an enzyme necessary to cleave a compound added to the agar (X-gal), thus, gives a blue pigment. On the other hand, colonies are white for those containing the plasmids with DNA inserts because they do not produce -galactosidase, therefore cannot cleave X-gal (Figure 2).
The following experiment outlines the construction of recombinant protein production in E.coli strain BL21 by using a bacterial plasmid vector pUC18/19 expressing Green Fluorescent Protein (GFP) to act as a recombinant protein product with the benefits of being easy to visualise and measure.
Figure 2. White and blue colonies.
MATERIALS AND METHODS
The experiment was carried out using the following materials and equipment: 2Âµl EcoRI/HindIII cut and cleaned PUC19 vector, 5Âµl EcoRI/HindIII cut and cleaned GFP insert, 2Âµl 10xT4 ligase buffer, 2Âµl T4 ligase(0.5 U ml-1) , and 9Âµl sterile water (H2O) ]to make up to 20Âµl volume[ .
100Âµl of competent BL21 E.coli cells on ice, 42Â°C water bath, ice bucket with ice, selective media plates (1.5% Luria Broth (LB) Agar, 40Âµg mL-1 X-gal, .1 mM IPTG, 50Âµg mL-1 ampicillin), sterile tubes, shaking incubator, spectrophotometer or similar device to measure optical density of the bacterial cultures, flasks, microcentrifuge.
Methods can be divided into four stages:
Ligation Reaction Stage:
In this stage, 2Âµl EcoRI/HindIII cut and cleaned PUC19 vector, 5Âµl EcoRI/HindIII cut and cleaned GFP insert, 2Âµl 10xT4 ligase buffer, 2Âµl T4 ligase (0.5 U ml-1) , and 9Âµl sterile water (H2O) were mixed and kept at room temperature for at least 30 minutes.
Transformation of ligation into cloning host stage: This stage was conducted by deforesting 100Âµl of competent BL21 E.coli cells on ice (with caution: do not allow warming at room temperature because the cells easily die), then adding 10Âµl of the ligation reaction from the first stage to BL21 E.coli cells. After which, these were incubated for up to 30 minutes on ice. After 30 minutes of incubation, the transformation mixture were taken out of the ice, heated in water bath at 42 Â°C for almost 75 seconds, and then followed by returning immediately into ice for a minimum of 2 minutes. After this, the cells were plated out on selective media plates (1.5% Luria Broth (LB) Agar, 40Âµg mL-1 X-gal, 10 Âµg IPTG, 50Âµg mL-1 ampicillin). Lastly, the plates were incubated at 37 Â°C for about 12-18 hours to allow growth of the organism.
Picking of colonies for the protein expression stage: 2x5ml LB +50Âµg ml-1 ampicillin in 30ml sterile tubes were prepared, and then 1xBlue individual colony and 1x white individual colony were selected and inoculated in separate tubes. After which, the tubes were incubated in a shaking incubator throughout the night at 37 Â°C with 220rpm speed.
Subculture and Growth of Recombinant E.coli for Protein expression: At this stage, 2x60ml sterile Luria-Bertani (LB), in 250ml conical flask were warmed, (1 per inoculums ) at 37 Â°C. Aseptically, the ampicillin was added to a last concentration of 50Âµg ml-1 ampicillin. Next, 1 ml of media was removed and was put in a cuvette to act as blank (one blank is enough for both), followed by addition of 600Âµl overnight to culture each individual colony to separate flask (1:100 inoculum). Then, the flasks were put back to the shaking incubator and incubated at 37Â°C with a speed of 200rpm. After that, a blank spectrophotometer was placed against media at 600nm and after 45 minutes, the samples were removed aseptically from flasks. After this, 1x1mL was removed from every flask and added to a fresh clean cuvette (take to next step 8). 1x1ml was then added to clean a Eppendorf tube. The OD600nm of culture in cuvette was measured and the result of growth curve was recorded (once the culture has reached an OD 600nm of 0.5, IPTG was added to final concentration 1Mm stock solution. Then, samples were spun down in the Eppendorf tube at the microcentrifuge maximum speed for 5 minutes (ensuring centrifuge is balanced before spinning). The supernatant was then removed and the pellet was suspended in 200Âµl Cell lysis buffer (10mMl Tris PH8.0, 300Mm NaCl , 10mg ml-1 Lysozyme). Resuspended cells were frozen at -20 c to the next day. Lastly, sampling was continued until OD600nm is no longer rising for two successive samples or until 16:30 pm.
RESULTS AND DISCUSSION
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Although it is supposed to harvest between 30-300 colonies per plate (210- 2100 colonies for all groups), just three blue colonies were observed in the plates between all groups, which means that the target protein (GFP protein) was not expressed efficiently in BL21 E.coli cells due to some factors influencing the expression level or to some technical problems during the experiment which will be discussed further.
The most popular strain, BL21 and its derivatives, which are good producing protein descended from E.coli B and thus is deficient in the Lon protease. Additionally, the BL21 background lacks the OmpT outer membrane protease. For expression work, BL21 cells should be taken from stock cultures that performed from fresh transforms. This step is crucial to ensure that the clone does not change and that each expression run gives optimal performance.
Transformation frequency is affected by DNA purity, how the cells are handled, and how the transformation was actually performed. DNA purification can however be done after digestion with restriction enzymes and after sequencing reaction. In addition, the most common mistake when transforming E. coli is by putting a lot of ligation mixture in the transformation.
Other factors that affect transformation with BL21 are the handling and the storage of the competent cells. Competent cells need to be preserved at -70Â°C to keep them at the peak. It is worthy of noting that 5-10-fold of efficiency usually is lost when tubes are placed back in the box and put back in the freezer. Moreover, cells must be thawed on ice, and the transformation should be started immediately after the cells are thawed. Incubating on ice is necessary for chemically competent cells. If heat shocked right away, the efficiencies will be down 10-fold. If incubated for only 15 minutes, it will be down 3-fold. In addition, time of heat shock (75 seconds) could not be enough, thus, is not efficient enough to transform E. coli. Moreover, water bath temperature may be not equilibrated (less than 42Â°C or a higher which decrease in transformation efficiency (Smith et al. 1992).
Also, the concentration of DNA has significant effect on the transformation efficiency. Usually, less amount of DNA is used. If DNA quantity is high, the result is fewer colonies because the impurities in the DNA will inhibit some of the cells from being transformed. Three factors are considered during induction conditions: (a) vector; (b) host strain; and (c) growth conditions. All these create a big impact on the expressing the target protein.
First on the list of considerations is the vector that is used to express GFP protein. The first thing that should be considered after cloning is to be sure that the sequence of the target protein is still accurate. Therefore, there should be a copy of the cloned plasmid's sequence before the experiment is done. Doing this, it will be known whether the sequence inserted into the expression vector is still right and in frame. This should really be considered because if there be any mutations, even a few mutated bases can have a serious effect on the expressed protein. Also, existence of rare codons should be checked for it can cause the resultant protein to be not functional or truncated. A sequence result of a few rare codons is still okay but when it is observed that a number of rare codons are present in a row, it can affect the expression a lot. Further, it is important to note the GC base concentration in the sequence. A high GC concentration can affect stability of the mRNA preventing successful translation causing the non-functionality of the protein.
After verifying the plasmid's sequence, the next to consider is the bacterial host being used. As mentioned, several hosts excel in producing different proteins. To exemplify, in a certain protein potentially causing genomic rearrangement, what is aimed for is a tight control over protein induction. However, there can be leaked expressions which affect the expression of the protein in the first place, affecting the cell's growth. Say for example when the system T7 Polymerase is used, then, it is needed to look for a host which contains the pLysS plasmid which encodes T7 lysozome. This will suppress the polymerase (T7) that could eventually reduce the background expression level. In cases like this, modification and change of hosts is suggested, allowing the expression of the correct protein.
The last factor to note is the protein's growth conditions. Here, the running an expression time course has to be considered. A fresh colony must be used to start and is then grown to stationary phase. Next is diluting the culture and allow growing to mid log, adding inducer and monitoring samples hourly. All these require proper timing as well as temperature. Also, choosing the concentration of the inducer (IPTG) is needed as this can also be toxic to the cells. Fresh inducers are also preferred.
And of course, all these factors will be known to be effective through a series on experimentation.
Definition of Terms:
Transformation efficiency is a measure of the ability of cells to be transformed. This is expressed as the number of transforms per microgram of pUC19 by using the following formula:
Colonies on plate / ng of control DNA X 1000ng/Âµg = (transformation (T) / Âµg plasmid DNA)
100 Î¼L equivalent to 0.01 ng DNA in the plate.
Growth curve (in Biology) is aÂ curveÂ in a graph showing the change in the number of cells in an experimental culture at different times. Normally, growth curve show S- shaped when plotted in log linear format (Figure 3) which is divided into four components: (1) Lag phase - the initial period when no increase in cell number is seen; (2) Log phase - when cells are growing at the maximum rate; (3) Stationary phase - growth decreases as a nutrient are depleted and waste products accumulate; and (4) Death phase - this is the result of prolonged starvation and toxicity.
Figure 4. Normal growth curve.
In this experiment, the growth curve showed same shaped as normal one (Figure 4)
Figure 5. Growth curve for this experiment.
The experiment was done to express protein of interest, the Green Fluorescent Protein (GFP). However, due to some errors in the protocols from ligation to induction or subcultring, the process resulted in an unfortunate outcome.
Escherichia coli are one of the most widely used hosts for the production of heterologous proteins and its genetics are far better characterized than those of any other microorganism. Recent progress in the fundamental understanding of transcription, translation, and protein folding in E. coli, together with serendipitous discoveries and the availability of improved genetic tools are making this bacterium more valuable than ever for the expression of complex eukaryotic proteins.
This study suggests consideration of the factors explained previously to successfully express GFP.
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