This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
In 1973 Stanley Cohen and Herbert Boyer pioneered the use of recombinant DNA technology for cloning and expression of genes in foreign organisms. They cloned DNA from the Salmonella typhimurium streptomycin resistance plasmid RSF1010 into the Escherichia coli plasmid pSC101 and observed tolerance to streptomycin among the transformants (Cohen et al., 1973). The first reported production of a human recombinant protein took place a few years later when the then newly started biotech company Genentech announced that they had managed to express the gene encoding human somatostatin in E. coli (Itakura et al., 1977). The value of the resulting bioactive substance was similar to that of somatostatin extracted from the brains of 500.000 sheep. In 1982 Genentech followed up this success with the product humulin, a recombinant insulin produced in E.coli and the first recombinant biotech drug to be accepted for market by the Food and Drug Administration. Today the production of recombinant proteins has become a huge global industry with an annual market volume exceeding $50 billion (Schmidt, 2004). At the start of the recombinant protein expression era the bacteria Escherichia coli and Bacillus spp. dominated as hosts for recombinant expression, but the realization that a protein may require a specific host physiology and biochemistry for optimal production stimulated a search for new hosts, both prokaryotic and eukaryotic. Parallel to this quest, recombinant DNA technology advanced tremendously thereby opening up possibilities for the use of novel organisms. As a consequence, many different expression systems for use in many different hosts are now available, including systems for use in yeasts (Gellissen et al., 2005), filamentous fungi (Nevalainen et al., 2005), insect and animal cell cultures (Wurm, 2004; Kost et al., 2005), gram-positive bacteria like Bacillus (Westers et al., 2004) and Streptomyces (Binnie et al., 1997), and gram-negative bacteria like Escherichia coli
Bacterial expression systems are the preferred choice for production of many prokaryotic and eukaryotic proteins. The reasons for this lie in the cost-effectiveness of bacteria, their well-characterized genetics, and the availability of many different bacterial expression systems. Among the hosts available for recombinant expression, Escherichia coli is in an exceptional position. This stems from the many decades of intense researchon its genetics as well as the broad scope of biotechnological tools available for genetic engineering of this organism. As a host for recombinant expression, E.coli is especially valued because of its rapid growth rate, capacity for continuous fermentation, low media costs and achievable high expression levels (Yin et al., 2007). One consequence of this popularity is that about 80% of all proteins used to solve three-dimensional structures submitted to the protein data bank (PDB) in 2003 were prepared in E.coli (Sørensen and Mortensen, 2005) and during 2003 and 2006, nine out of 31 approved therapeutic proteins were produced in E.coli (Walsh, 2006), among them important growth factors, insulins and interferons (Schmidt, 2004).
Green Fluorescent Protein (GFP) was isolated from the jellyfish Aequorea aequorea in 1962 (Shimomura et al., 1962) where it was found as a companion protein to aequorin, the well-known chemiluminescent protein of the same species. It was noticed that living A. aequorea tissue had an emission spectrum peaking at 508nm and looking green but pure aequorin peaked in the blue range, at 470nm (Tsien, 1998). This then led Shimomura's group to discover GFP and suggest radiation-less energy transfer as the mechanism for exciting the protein. Its structure has been determined to consist of an 11 stranded Î²-barrel containing the chromophore made up of a single Î± helix as shown in Figure1.
Figure 1. Protein structure of GFP (Anthony, 2012).
Its use as a tool in molecular biology was not realised until 1992 when Prasher reported the cloning and sequence of GFP (Prasher et al., 1992). Since 1994 GFP has been used as a reporter protein (Chalfie et al., 1994) flagging its own presence and therefore also proteins under the same control, by emitting green light (Î»em = 508 nm) upon excitation with near ultraviolet light (around 395 nm) or blue light (around 470 nm) (Ito et al, 1999). Since then many mutations have been developed looking to improve the emission or to focus it to a single wavelength (Heim et al., 1995) or to change the color of the emitted light itself.
Recombinant DNA molecules usually contain a DNA fragment inserted into a bacterial vector.
Polymerase chain reaction (PCR) , a speciï¬c gene or DNA region of interest is isolated and ampliï¬ed by DNA polymerase extracted from a heat-tolerant bacteria. PCR "ï¬nds" the DNA region of interest (called the target DNA) by the complementary binding of 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. Most cutting is done with the use of bacterial restriction enzymes. These enzymes cut at speciï¬c DNA sequences, called restriction sites, and this property is one of the key features that make restriction enzymes suitable for DNA manipulation. These enzymes are examples of endonucleases that cleave a phosphodiester bond (Anthony, 2012). The key property of some restriction enzymes is that they make "sticky ends. The restriction enzyme EcoRI (from E.coli) recognizes the following sequence of six nucleotide pairs in the DNA of any organism:
The enzyme EcoRI makes cuts only between the G and the A nucleotides on each strand of the palindrome (Figure.2).
Figure 2. Forming of recombinant DNA molecules. (Anthony, 2012).
The recombinant DNA molecules are transferred into bacterial cells, and, generally, only one recombinant molecule is taken up by each cell. The recombinant molecule is ampliï¬ed along with the vector during the division of the bacterial cell. This process results in a clone of identical cells, each containing the recombinant DNA molecule, and so this technique of ampliï¬cation is called DNA cloning. The next stage is to ï¬nd the rare clone containing the DNA of interest.
Bacterial plasmids (vectors) are small circular DNA molecules that replicate their DNA independent of the bacterial chromosome. The plasmids routinely used as vectors carry a gene for drug resistance and a gene to distinguish plasmids with and without DNA inserts. These drug-resistance genes provide a convenient way to select for bacterial cells transformed by plasmids: those cells still alive after exposure to the drug must carry the plasmid vectors. However, not all the plasmids in these transformed cells will contain DNA inserts. For this reason, it is desirable to be able to identify bacterial colonies with plasmids containing DNA inserts. Such a feature is part of the pUC18 (or pUC19) plasmid vector shown in Figure 2; DNA inserts disrupt a gene (lacZ) in the plasmid that encodes an enzyme (-galactosidase) necessary to cleave a compound added to the agar (X-gal) so that it produces a blue pigment. Thus, the colonies that contain the plasmids with the DNA insert will be white rather than blue (they cannot cleave X-gal because they do not produce -galactosidase).
Figure 3. Use of a plasmid vector, pUC18/19.
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.
Materials and Methods
The experiment was carried out using the following materials and Equipments: 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.
It can be divided into three 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) are mixed and kept at room temperature for at least 30 minutes.
Transformation of ligation into cloning host stage: this stage conducted by deforesting 100Âµl of competent BL21 E.coli cells on ice (with caution do not allow to warm to room temperature), then adding 10Âµl of the ligation reaction from the first stage to BL21 E.coli cells. They are then incubated for up to 30 minutes on ice. Next step, is done by taking out the transformation mixture out of the ice and heated in water bath at 42 Â°C for almost 75 seconds, then followed by return immediately into ice for a minimum of 2 mins. Then the cells were plated out on selective media plates (1.5% Luria broth (LB) Agar, 40Âµg mL-1 X-gal, .1 mM IPTG, 50Âµg mL-1 ampicillin). Lastly, the transformation mixture is incubated at 37 Â°C for 12-18 hours afterdriedd.
Picking of colonies for the protein expression stage: 2x5ml LB +50Âµg ml-1 ampicillin in 30ml sterile tubes were prepared, then 1xBlue individual colony and 1x white individual colony selected and inoculated in separate tubes. Then the tubes were incubated with shaking incubator throughout the night at 37 Â°C , speed: 220rpm.
Subculture and Growth of Recombinant E.coli for Protein expression: At the beginning, 2x60ml sterile Luria-Bertani (LB), in 250ml conical flask were warmed , (1 per inoculums ) at 37 Â°C, Then 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 ouh), followed by addition of 600Âµl overnight to calture of each individual colony to separate flask (1:100 inoculum), the flasks were put back to the shaking incubator and incubated at 37Â°C, speed: 200rpm , after that blank spectrophotometer was placed against media at 600nm , after 45 minutes the samples were removed aseptically from flasks, then from every flask 1x 1mL was removed and added to a fresh clean cuvette (take to next step 8) and 1x1ml was added to clean Eppendrof (take to step 9) . 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 Eeppendrof tube at max speed in Microcentrifuge for 5 minutes , ensure centrifuge is balanced before spinning , the supernatant was removed and pellet ,then 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
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 plates between all groups, which mean that protein of interest (GFP protein ) was not expressed (inefficient) in BL21 E.coli cells due to some factors influenced the expression level or to some technical problems during the experiment which will be discussed.
The most popular strain, BL21 and its derivatives, which are good producing protein, are 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 insure that the clone does not change and that each expression run gives optimal performance.
Transformation frequency is affected by the purity of the DNA, how the cells are handled, and how the transformation was actually performed. In the impurities in the DNA usually spin columns can be used to purify DNA from PCR reactions, ligations, endonuclease digestions, or other treatments. In addition, the most common mistake when transforming E.coli is to put a lot of ligation mixture in the transformation.
Other factors that effect transformation with BL21 are the handling and the storage of the competent cells. Competent cells need to be reserved at -70Â°C to keep them at the peak .It is worthy of noting that 5-10-fold of efficiency usually lost if tube put back in the box and place 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 you heat shock right away, the efficiencies will be down 10-fold. If incubate for only 15 minutes, it will be down 3-fold. In addition, time of heat shock (75 second) could be not enough , thus, affect the efficiency enough to transformation of 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 using more, the result is fewer colonies because the impurities in the DNA will inhibit some of the cells from being transformed.
There are main factors to consider during induction conditions: Vector, Host Strain, and Growth Conditions. These three factors have the biggest impact on the expression of the protein of interest. First on the list of considerations is the vector that is used to express GFP protein. The first thing should be considered after cloning, the protein of interest is still in frame. It is recommended that before any experiment is carried out the first thing is should be done is cloned plasmid (or a few different clones) sequenced. This will show if the sequence you inserted into the expression vector is still correct and is still in frame. This is especially important if the construct contains any PCR fragments. If there are any point mutations or the sequence gets out of frame by even a few bases it can have dramatic effects on the protein that expressed. Another thing to check before expressing is if the GFP protein sequence contains long stretches of rare codons. This can cause the protein that is expressed to be truncated or non-functional. A few rare cordons spread around the protein are OK in most cases, but if there are a number of rare codons in a row, then it can have a big effect. The third sequence related step to optimize the protein production is to make sure there is not a high GC concentration at the 5' end of GFP protein. This could potentially cause problems with the mRNA's stability, and could prevent it from being translated correctly, which would also lead to truncated or non-functional proteins. If your sequence is GC heavy at this end, you can try to make a few silent mutations to break up long stretches to try and help stability.
After the plasmid is sequence verified, the next factor is the bacterial host that is used. There are almost as may hosts as there are expression vectors, with certain hosts excelling in producing different types of proteins. For example if you have a toxic protein, or a protein that could potentially cause genomic rearrangement, you will want a vector that gives you very tight control over the induction of your protein. There can be "leaky" expression (i.e. expression of your protein without the addition of your inducer) that can potentially have adverse effects on the cells growth or even prevent your cells from over-expressing your protein in the first place. If you're utilizing the T7 polymerase system, then look for a host containing the pLysS plasmid, as this will code for T7 lysozyme, which will suppress the T7 polymerase and can greatly reduce the level of background expression. If as stated before you have a protein that contains a large number of rare codons, then look for a host with the genes for the necessary tRNA's already present, which should allow your protein to express correctly. Sometimes simply changing hosts can have a dramatic effect on the amount of protein produced and the stability of the protein that is made, so if one host isn't giving you the results you need, then feel free to switch your host up.
The third and final factor to consider when expressing a protein is growth conditions. When first starting out with the protein induction it is very important to run an expression time course, where you take a fresh colony from a streaked plate, and grow the culture to stationary phase. Next, dilute the overnight culture 1/100 and grow to mid log phase, then add the inducer and induce your protein for a number of hours, taking 1mL samples every hour or so. Once these samples are lysed, you can run an SDS-PAGE gel to determine your protein production levels. You might get great induction the first time, or you may have to tweak your conditions in order to get really good expression levels. Other factors that may need to be controlled for are the bacterial growth rate (determined by taking OD measurements during the induction process), and the temperature during induction. Some constructs will express perfectly fine at 37Â°C, while others need to be bumped down to 30Â°C to induce correctly. The concentration of the inducer too will have an effect, as many inducers (IPTG) can be toxic to the cells that they are inducing. Using freshly made inducer is good step to making sure you always have consistent results. Only through experimentation can you determine what will be best for your construct, and give you the most robust expression levels.
Transformation efficiency is a measure of the ability of cells to be transformed. Transformation efficiency 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.
In general growth curve shows the S- shaped when plotted in log linear format as shown in figure 4, that separated into four phases:
C:\Users\Mazen\Desktop\picture protein 2.jpg
Figure 4. Population growth curve for cells in liquid medium (Reed et al, 1998).
Lag phase ; the initial period when no increase in cell number is seen.
Log phase; when cells are growing at the maximumm 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 , growth curve presented same shaped as normal one as shown in figure 5.
Figure 5. Represent both growth curves for Blue and White colonies in culture
The main goal for the experiment was to express the protein of interest (GFP). However, factors influencing transformation efficiency include technique errors, the temperature and length of the incubation period, the growth stage of the cells, and using the correct mass of plasmid DNA. Escherichia coli is one of the most important hosts in modern day recombinant protein production. Throughout academia and industry its uses are widespread and with sequence data available for some of the most common strains of the bacteria it has been a favourite organism for many metabolic engineering and metabolic modelling projects in the past (Berry, 1996; Koffas et al., 1999).