Protein Expression - Lab Report Example

? Lab Report on Protein expression Introduction: Green Fluorescent Protein, a molecule that produces the greenish glow in luminescent jellyfish, remains a popular protein today when it comes to protein expression. The principal behind the glow entail energy transfer that results in a change of the shape or conformation of that protein resulting in production of light plus colour (luminescence). Moreover, the GFP (Green Fluorescent Protein) gene usually exists naturally in the form of specialized photogenic cells found in the jellyfish Aquorea Victoria umbrella. This fluorescent protein can also be expressed in the bacteria, Escherichia coli. When the fluorescent protein gets exposed to U.V light source of long wave band, it produces a green light. Therefore, in some cases, the given GFP gene can get attached to the given end of a gene required to encode a given protein of interest. Cloning Cloning by definition involve making an identical copy of the original. Cloning is usually considered as an amplification process, since it entails making of identical copies from a single copy. Therefore, cloning DNA simply involves recombinant DNA. Often, there exits two different DNAs which are required in the process of cloning. One of them include an insert (the given molecule to be cloned), then secondly, a vector (cloning vehicle). In addition, a white/blue selection vector usually remains as the common plasmid cloning vector type. The vector contains an original of replication, allowing plasmid replication to occur, hence providing the actual amplification step that allows selection of a given bacteria which contain the vector. Furthermore, it contains the E.coli lacZ gene that allows for selection and insertion of DNA fragment into a given vector. Therefore, the lacZ gene contains an MCS (multiple cloning site), an oligonucleotide sequence that exists in series with other restriction endonuclease recognition sites which are usually arranged in tandem providing the same reading frame like the lacZ gene. Therefore, this is surely the heart around white/blue selection. Usually, the lacZ gene often codes for (B-gal) B-galactosidase, a given enzyme which cleaves lactose galactoside bond. Furthermore, it also can cleave galactoside in X-gal, an artificial subtract. X-gal, if added in a media containing growing bacteria, usually turns blue when cleaved. When a DNA fragment gets inserted into the MCS, usually the lacZ gene becomes disrupted, resulting to its inactivation, leading to B-gal not able to cleave X-gal, causing presence of white colonies. Vector and the insert often get cut by the same destruction enzyme leading to production of single-stranded sticky ends. In this case, the variable entails the ability for it to select various enzymes which recognize difference restriction sites. After this, ligation is performed to generate fewer multimers. Next, entail the transformation process of which involve DNA introduction into a given host cell. In E.coli, DNA has to be forced in it since it does not undergo a normal physical transformation. Usually, two methods can be used for transformation: on entails washing the given bacteria with very high salt concentration of calcium chloride, or by passing current through it (electroporation). E.coli contains lacZ gene hence function with the white/blue system. The commonly used strain is DH5 alpha. Usually, transformation produces multiple bacteria types, some of which are unwanted since they contain vectors without inserts. Then, the transformed bacteria are usually placed in an agar plate which contains ampicillin and IPGT (Isopropyl Thiogalactoside), a B-gal inducer. Ampicillin hence kills bacteria which would not have transformed. The remaining population usually thrive to produce white colonies and blue colonies. It is necessary to remove false positive clones. Materials and Methods: Materials: The following materials were required for the experiment to be successful. 2µl HindIII/ EcoRI cut plus cleaned PUC19 vector 5µl HindIII/ EcoRI/ cut plus cleaned GFP insert Ligase2µl T4 (0.5 U ml-1) Ligase buffer 2µl 10xT4 Sterile H2O for making 20µl volume Methods: The ligation reaction: It is done through mixing the above materials to a concentration of 20µl, then incubating the ligation reaction mixture at a room temperature for thirty minutes. Transformation of ligation into cloning host: 100µl of BL21 E.coli becomes defrosted on ice, then 10µl of the set ligation reaction added to it, then incubated on ice for approximately 30 minutes. Then transformation mixture is taken out of the ice, then heat to 42 degrees Celsius in 75 seconds. Then transformation mixture is taken out of ice and heat shock at 42 °C for 75 seconds (heat shocking). After this, it is immediately taken back to ice for 2 minutes. Then cells are plate out on selective media plates (4oµg ml-1 x-gal, 50µg ml-1 ampicillin, 1.5 % LB agar and 0.1mM IPTG. Transformation then allowed to dry out on the given plates then incubated for 12-18 hours at 37 degrees Celsius. Picking of colonies for protein expression: 50µg ml-1 ampicillin plus 2x5ml LB + in 30ml sterile tubes is set up. 1xBlue individual colony plus 1x white individual colony are picked up, and then inoculated in separate tubes. These are left in a shaking incubator at 37 degree Celsius overnight at 220rpm. Subculture and growth of recombinant E.coli for protein production: Firstly, 2x60ml sterile LB is pre-warmed in a 250ml conical flask at 37 degrees Celsius. Later, ampicillin is added to a concentration of 50µg ml, and then 1ml of media removed and placed in cuvette. These acts as blank. 600µl is added overnight to each individual colony culture to separate flasks (1:100 inoculums). Then, return flask placed in shaking incubator at 200rpm. Then, subsequently placed in blank spectrophotometer running at 600nm against the given media, then at forty five minutes, 1 ml of the given samples are removed from the flasks and added fresh clean cuvette and another to clean eppendrof. OD 600nm of culture then measures in cuvette, then results recorded to create a growth curve. .Once O.D 600nm of 0.5 reached, IPTG becomes added to 1Mm stock solution. Samples in eppendrof tube get spanned at maximum speed in micro centrifuge in approximately 5 minutes. Ensure to freeze re-suspended cells at negative 20 degrees Celsius in the following day. Freeze re-suspended cells at -20 c following day. Then, finally continue sampling till OD600nm no longer rise 16:30 or for two successive samples. Results: Times (mint) Blue colonies White colonies 45 mint 0.155 0.184 90 mint 0.567 0.610 135 mint 1.282 1.325 180 mint 1.771 1.801 225 mint 1.987 2.005 270 mint 2.161 2.148 Table showing difference in absorbance between the blue colonies and white colonies during various periods of induction FIGURE 2: The curve illustrates growth rate of non-recombinant (blue) and recombinant (white) in E.coli. Discussion The process involved in recombinant protein expression is sometimes considered inefficient since it got a number of steps presenting with technical erros. A good exampe entail ligation volume of sterile water used. The original test requires 9 ?m of sterile water added to have ligation reaction mixture, but other students, increased the concentration because of the instructions in the protocol. This hence affected the transformation efficiency of BL21 strain. This is probably has affected the efficiency of transformation of BL21 strain .In E.coli transformation, it is a mistake adding too much ligation mixture. In transformation stage, there are so many factors that might affect its efficiency. One of them is whether the chosen BL21 bacteria remains suitable or not for the experiment. Secondly, whether the temperature used is too low or too much so that they may have detrimental effect on the outcome. Another important factor here is the exact time of transformation might not have been measured precisely in the experiment. In addition, transformation frequency usually affects the purity of a given DNA as it dates back to how cells get handled. In IPTG induction, errors might have resulted due to inducers not properly preserved or not fresh. Induction that is poor usually result due to a number of reasons, some of which include plasmid loss because of instability that result by expression of toxic proteins or poorly translated RNA or unstable mRNa which are very rare in E.coli. In figure 2, a bacterial growth curve is displayed showing the four phase characteristic. It starts with an initial lag phase as it depicts a period where bacteria adapt to the fresh medium. Then, log phase follows, here growth is exponential. Phase 3 is stationary phase, nutrients here are becoming limited with an existed high population existing. Here multiplication rate is equivalent to death rate. Last phase is decline phase and denotes higher death rate compared to multiplication rate due to depleted nutrients. Conclusion In summary, protein expression is achievable by DNA recombination. With the help of E.coli, DNA recombination was made possible through cloning of various strains with specific colony characteristics. Hence, gene cloning is achievable. Read More