Construction of Recombinant DNA in E Coli

Construction of Recombinant deoxyribonucleic acid in E ColiIn 1973 Stanley Cohen and Herbert Boyer pioneered the use of recombinant deoxyribonucleic acid technology for re-create and musing of genes in foreign organisms. They cloned desoxyribonucleic acid from the Salmonella typhimurium streptomycin resistance plasmid desoxyribonucleic acid 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 excessivelyk send off a few years later when the thusly newly inaugurationed 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 bio moive nerve center 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 i n E.coli and the first recombinant biotech drug to be accepted for market by the Food and do drugs Administration. Today the production of recombinant proteins has become a huge global industry with an annual market volume exceeding $50 billion (Schmidt, two hundred4). At the start of the recombinant protein expression era the bacteria Escherichia coli and Bacillus spp. dominated as entertains for recombinant expression, but the realization that a protein may require a specific host physiology and biochemistry for optimum production stimulated a search for new hosts, both prokaryotic and eukaryotic. Par every(prenominal)el to this quest, recombinant DNA technology advanced tremendously thereby opening up possibilities for the use of novel organisms. As a conchronological succession, many unalike expression systems for use in many different hosts argon now available, including systems for use in yeasts (Gellissen et al., 2005), filamentous fungi (Nevalainen et al., 2005), insect a nd 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 coliBacterial expression systems atomic subject 18 the preferred choice for production of many prokaryotic and eukaryotic proteins. The reasons for this lie in the cost- gistiveness 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 cons equence 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 (Srensen 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 actors, 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 detect 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 indeed led Shimomuras multitude to discover GFP and suggest radiation-less energy transfer as the mechanism for exciting the protein. Its structure has been designated to consist of an 11 s tranded -barrel containing the chromophore made up of a single helix as shown in Figure1.Its use as a tool in molecular biology was not realised until 1992 when Prasher reported the re-create 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 experience, by emitting green glitter (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 wave space (Heim et al., 1995) or to change the color of the emitted light itself.Recombinant DNA molecules unremarkably 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 a much smaller size to begin with they can be inserted into a vector. Most cutting is make with the use of bacterial restriction enzymes. These enzymes cut at specific DNA sequences, called restriction sites, and this property is one of the central features that lease restriction enzymes suitable for DNA manipulation. These enzymes are examples of endonucleases that split 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 organism5-GAATTC-33-CTTAAG-5The enzyme EcoRI makes cuts merely between the G and the A nucleotides on each strand of the palindrome (Figure.2).The recombinant DN A molecules are transferred into bacterial cells, and, generally, that one recombinant molecule is interpreted 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 proficiency of amplification is called DNA cloning. The succeeding(prenominal) 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 try 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 en trust 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 vex 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).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 MethodsMaterialsThe experiment was carried out using the following materials and Equipments 2l EcoRI/HindIII cut and cleaned PUC19 vector, 5l EcoRI/HindIII cut and cleaned GFP insert, 2l 10xT4 ligase buffer, 2l T4 ligase(0.5 U ml-1) , and 9l sterile water (H2O) to make up to 20l volume . atomic number 6l of competent BL21 E.coli cells on ice, 42C water bath, Ice bucket with ice, selective media plates (1.5% Luria broth (LB) Agar, 40g mL-1 X-gal, .1 mM IPTG, 50g mL-1 ampicillin), sterile tubes, shaking incubator, Spectrophotometer or similar device to measure optical density of the bacterial cultures, flasks, Microcentrifuge.MethodsIt can be divided into three stagesLigation Reaction stage in this stage 2l EcoRI/HindIII cut and cleaned PUC19 vector, 5l EcoRI/HindIII cut and cleaned GFP insert, 2l 10xT4 ligase buffer, 2l T4 ligase (0.5 U ml-1) , and 9l sterile water (H2O) are mixed and kept at room temperature for at least 30 minutes. version of ligation into cloning host stage this stage conducted by deforesting 100l of competent BL21 E.coli cells on ice (with caution do not allow to firm to room temperat ure), then adding 10l 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 displacement mixture out of the ice and heated in water bath at 42 C for well-nigh 75 seconds, then followed by return direct into ice for a minimum of 2 mins. Then the cells were plated out on selective media plates (1.5% Luria broth (LB) Agar, 40g mL-1 X-gal, .1 mM IPTG, 50g 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 +50g 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 passim the night at 37C , revivify 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 C, Then aseptically the ampicillin was added to a last stringency of 50g ml-1 ampicillin. Next 1 ml of media was remote and was put in a cuvette to act as blank (one blank is enough for both ouh), followed by addition of 600l overnight to calture of each individual colony to separate flask (1100 inoculum), the flasks were put back to the shaking incubator and incubated at 37C, 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 new 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 Eepp endrof tube at max speed in Microcentrifuge for 5 minutes , ensure centrifuge is balanced in the lead spinning , the supernatant was removed and pellet ,then the pellet was suspended in 200l 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 1630 pm.Results and discussionAlthough 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 unspoilt 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 bear upon 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 -70C 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 42C 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 thre e 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 mRNAs 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 le aky 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 youre utilizing the T7 polymerase system, then look for a host containing the pLysS plasmid, as this will code for T7 lysozyme, which will curtail 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 tRNAs 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 isnt 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 fi rst 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 37C, while others need to be bumped down to 30C to induce correctly. The concentration of the inducer too will have an effect, as many inducers (IPTG) can be to xic to the cells that they are inducing. Using freshly made inducer is good step to make sure you always have consistent results. Only through experimentation can you determine what will be best for your construct, and give you the most strong expression levels.Transformation efficiencyTransformation efficiency is a measure of the ability of cells to be transformed. Transformation efficiency is expressed as the number of transforms per mcg of pUC19.By using the following formulaColonies on plate / ng of control DNA X 1000ng/g = (transformation (T) / g plasmid DNA)100 L equivalent to 0.01 ng DNA in the plate.Growth curveIn general growth curve shows the S- shaped when plotted in log unidimensional format as shown in figure 4, that separated into four phasesLag 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. ending phase this is the result of prolonged starvation and toxicity.ConclusionThe 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).