The Use Of Recombinant Dna Technology

Published Date: 02 Nov 2017

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 β-barrel containing the chromophore made up of a single α helix as shown in Figure1.

Figure 1) Protein structure of GFP (Source: Protein data bank).

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 (λem = 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 colour of the emitted light itself.

Recombinant DNA molecules usually contain a DNA fragment inserted into a bacterial vector.

Polymerase chain reaction (PCR) , a specific gene or DNA region of interest is isolated and amplified by DNA polymerase extracted from a heat-tolerant bacteria. PCR "finds" the DNA region of interest (called the target DNA) by the complementary binding of specific short primers to the ends of that sequence.The long chromosome-size DNA molecules of genomic DNA must be cut into fragments of 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 specific 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. 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:

5-GAATTC-3

3-CTTAAG-5

The enzyme EcoRI makes cuts only between the G and the A nucleotides on each strand of the palindrome (Fig.1).

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 amplified 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 amplification is called DNA cloning. The next stage is to find 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 10-9; 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).

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The following experiment outlines the construction of recombinant protein production in E.coli strain BL21by 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

Materials:

The experiment was carried out using the following materials and Equipments: 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) , and 9µl sterile water (H2O) ]to make up to 20µl volume[ .

100µl of competent BL21 E.coli cells on ice, 42°C water bath, Ice bucket with ice, selective media plates (1.5% Luria broth (LB) Agar, 40µg mL-1 X-gal, .1 mM IPTG, 50µg mL-1 ampicillin), sterile tubes, shaking incubator, Spectrophotometer or similar device to measure optical density of the bacterial cultures, flasks, Microcentrifuge.

Methods:

It can be divided into three stages:

Ligation Reaction stage: in this stage 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) , and 9µl 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 100µl of competent BL21 E.coli cells on ice (with caution do not allow to warm to room temperature), then adding 10µl 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 °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, 40µg mL-1 X-gal, .1 mM IPTG, 50µg mL-1 ampicillin). Lastly, the transformation mixture is incubated at 37 °C for 12-18 hours afterdriedd.

Picking of colonies for the protein expression stage: 2x5ml LB +50µg 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 °C , speed: 220rpm.

Subculture and Growth of Recombinant E.coli for Protein expression: At the beginning, 2x60ml sterile LB, in 250ml conical flask were warmed ,(1 per inoculums )at 37 °C, Then aseptically the ampicillin was added to a last concentration of 50µg 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 600µl 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 37°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 200µl Cell lysis buffer (10mMl Tris PH8.0, 300Mm Nacl , 10mg ml-1 Lysozyme). resuspended cells were freezes at -20 c to the next day.Lastly, sampling was continued until OD600nm has no longer risen for two successive sample or until 16:30 pm.