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The week 1 activity helped to understood cell lysis technique using SDS like detergent to disrupt the cell membrane. In this regard, E.coli has been chosen to understand cell lysis as well as the separation of plasmid DNA. Along with that sodium hydroxide was used to denature as well as separate genetic material like DNA. Microcentrifugation is a useful technique to completely separate cell debris from DNA containing supernatant solution as well as purification of DNA. Agarose gel electrophoresis using TAE buffer is useful to separate negatively charged DNA according to its size and mass. The importance of competent cells was learned this week along with the procedure of converting normal cells into competent cells. Other than that the procedure for the transformation of plasmid DNA into these competent cells was also taught. Preparation of plates using ampicillin antibiotics in the media ensures conversion of bacterial cell that contain targeted DNA that does not restrict the growth of these cells or kill them even in the antibiotics. In Week 2, purification of Taq polymerase was taught so that more of these enzymes can be produced in the lab. Taq polymerase is useful for PCR or Polymerase Chain Reaction that helps to produce two DNA from a single one. This enzyme is frequently used in medicine to detect any disease such as cancer. A comparative experiment was performed between commercial Taq polymerase and the purified ones. In the 3rd week, experiments were performed to improve the efficiency of purified Taq polymerase.
In this lab write up, at first results of different experiments performed in three weeks will be written. At first, an overview will be given on the result of cell lysis and centrifugation. After that, the result of agarose gel electrophoresis will be discussed. After that, a brief discussion will be done on the result of Taq polymerase isolation as well as purification. In addition, the result of week 3 experiments will be discussed to improve the capability of purified Taq polymerase. In the discussion part, plasmid isolation from E. coli will be reviewed at first. After that, a discussion will be done on the purification of host cells along with the purification of Taq polymerase from cell Debris. In this part, an elaborate discussion will be done about the procedure of preparing Taq polymerase in the laboratory along with its purification and isolation. A brief deliberation will be done on Polymerase chain reaction with the help of Taq polymerase. In the end, a brief description will be done of the overall experimental design using sufficient resources from other research papers written on the same experiments.
Week 1
The first week's task was to perform cell lysis to isolate the plasmid DNA. Microfluidic devices are used to perform this cell lysis. At first, the cell membrane is destroyed using a detergent that usually reacts with the cell membrane and creates pores on them that help to release the genetic materials like protein, DNA, as well as RNA (Shehadul Islam, Aryasomayajula & Selvaganapathy, 2017). With the help of microcentrifugation, cell debris is removed to get the supernatant plasmid solution.
After that “Agarose gel electrophoresis” is performed on the supernatant plasmid solution for separating the DNA fragments. At first, the agarose gel is prepared using TAE (Tris-acetate) buffer, completely cooled down before the addition of the supernatant. After that, the current is applied and the DNAs start migrating towards the positive electric field. The movement of the DNA from the negative end to the positive end gets captured in this step. The migration occurs due to the presence of a phosphate backbone of DNA which is negatively charged. After that preparation of competent cells of the E.Coli is done where at first, colonies are isolated and incubated at room temperature overnight. Following the second overnight incubation, the cells are pellet in cold condition and centrifuged. This procedure results in competent cells where bacterial colonies are immediately placed into tubes and stored in a chilled state (approximately -80°C). These competent cells are now ready to receive isolated plasmids that are further used in different biological applications.
Week 2
Taq bacterias that are thermostable at higher temperatures are isolated from the bacterial lysate. Taq polymerase collected from this bacterium is used for DNA replication due to its higher heat stability. Application of excessive heat denatures other bacteria other than Taq polymerase which are purified using centrifugation and results in “total purified Taq”, which are stored for later use. SDS-PAGE is run to separate Taq polymerase from the supernatant and is labelled as “purified Taq”.
After that protein estimation using BCA reagent is done to understand the total protein content or Taq polymerase content in this purified taq as well as commercial taq. The PCR (Polymerase Chain Reaction) setup is done to compare the DNA replication capability of both taq polymerase. This results in the formation of two new DNA from each strand. Hence, at the end of PCR, multiple copies of DNA are produced. In this case, the recombinant plasmid of E. coli produced in week 1 was used as the DNA template to produce multiple copies of recombinant DNAs.
A lab test was performed with 5 samples (1 Blank) and one unknown test sample for protein estimation using BCA. Three consecutive tests were done and the absorbancewas measured at 562 nm to understand the protein concentration in mg/ml. The graph shows the protein concentration in the unknown sample is between 0-0.5 mg/ml, according to the absorbance it showed.
Week 3
In week two the capability of commercial and purified Taq polymerase was determined. In this week several tests were performed to improve the efficacy of purified Taq polymerase. Ethanol precipitation was performed to locate the Taq in the pellet. In addition, Viva500 1000kDa was used to make the Taq polymerase more concentrated so that it can produce more DNA. Moreover, the treatment with DNase I was done to reduce the overactivity of DNA that might create problems during PCR. Therefore, all these activities result in a better outcome of PCR using purified Taq polymerase.
Plasmid isolation from E.coliCell lysis using detergent is done to get the genetic materials of the cell. In this case, E.coli bacteria have been chosen to separate the plasmid DNA to perform the PCR (Silva-Santos et al. 2017). However, the genetic material cannot be directly isolated from the cell until centrifugation is performed. The centrifugation technique uses a centrifugal force or a high-speed rotation where the cell debris gets separated in the lateral wall, away from the axis or centre. In addition, the clear supernatant solution, containing less dense material accumulated in the centre (Elbing & Brent, 2019). This supernatant solution contains the plasmid DNA which is used for various purposes. Additional centrifugation ensures no remaining cell debris and results in purified supernatant. This supernatant is now ready to undergo “Agarose gel electrophoresis”. This technique is important to isolate the DNA based on its size and mass and use them in forget steps (Green & Sambrook, 2019). The movement can be seen towards the positive electrode as the DNA is positively charged due to the presence of a phosphate backbone. Following staining, with DNA-binding dye, the DNA can be visible under UV rays and thus plasmid DNA gets separated from E.coli.
The ability of natural Host cells like yeast or bacteria to take genetic material naturally from an extracellular environment is significantly low. The reason why these cells have to get transformed into “competent cells”. Usually, Calcium chloride is used as a competent agent for E.coli. There is a lengthy procedure of inoculation, centrifugation, resuspension, and incubation of cells in specific parameters. Following the second centrifugation and resuspension in 0.5ml 0.1 M Calcium chloride at 0°C the normal host E.coli cells finally transform into competent cells that are ready to accept the genetic material. This step is called “Transformation” where the DNA from the donor bacteria gets bound to the competent cell and gets integrated into chromosomal DNA following a “Homologous Recombination”. In this way “Recombinant plasmid (P)” along with “Negative control (-ve)”, as well as “positive control (+ve)” can be transferred into the competent host cells of E.coli.
The lesson of week 1 was utilised in the week where the isolation of Taq polymerase was done from the bacterial lysate. Taq (Thermos Aquatequs) is a bacterium that cannot get denatured easily at higher temperatures and has thermal stability at 93-95°C. The importance of this enzyme is significant in PCR which is needed in gene therapy for treating any disease, helps in the identification of any genetic disorders, and even detection of diseases, including cancer. Following identification of this enzyme it has created a revolution in the medical field. Taq polymerase is easily available commercially but its isolation and purification in the laboratory can save a lot of expenses in the lab and the reason why it was a part of the week 2 lesson. At first, the bacterial lysate is centrifuged and heated at 75°C to ensure all the bacteria get denatured except the Taq. Being a thermostable bacterium it does not destroy at such a high temperature. Following another centrifugation, all the other bacteria get separated leaving only the Taq polymerase that will be used in the next step which is the PCR experiment (Fang et al. 2016). Thus, in simply 2-3 steps “total Purified Taq” can be prepared in the lab.
An additional step was performed using SDS-PAGE (Sodium-dodecyl sulphate-polyacrylamide gel) where SDS-buffer is mixed with the purified Taq labelled as “unpurified Taq” to perform SDS-PAGE electrophoresis to separate Taq polymerase according to the mass and size. SDS buffer is then again mixed with bacterial lysate and unpurified Taq and is mixed together. These are again denatured, centrifuged and run on SDS-PAGE. SDS is a detergent that has a b protein denaturing effect and breaks the polypeptide chain of the protein and makes it negatively charged due to the presence of sulphate in its structure. In this way, proteins get separated based on polypeptide chain length.
The BCA assay is performed after that to quantify the protein amount in the sample. Here BSA or (Bovine serum albumin) is used as the alkaline agent and in its presence, the protein can reduce cupric ion into a cuprous ion that changes the colour of the solution to change into purple. This happens due to the presence of bicinchoninic acid.
Taq polymerase is mainly used for doing PCR or replication of DNA. Denaturation of DNA takes place at 92-94°C, where two strands of DNA get separated. In the annealing step, which occurs at about 50-56°C, nucleotide primers get attached to the 3' end of the DNA strands (Green & Sambrook, 2019). The last step, which is an extension, occurs at 72°C, the Taq polymerase acts and adjoins nucleotides molecules to make new DNA strands. Every time these steps are repeated the DNA gets doubled each time.
DNase I am useful in destroying excess DNA that can cause an interruption in the function of Taq polymerase. Moreover, 55% ethanol can be used to precipitate the Taq polymerase and by using Viva 500 1000 KDA, the concentration of this enzyme can be enhanced to yield better Taq polymerase than the commercial one.
Inoculation in 100ml LB (Luria Bertani) media to ensure bacterial cell growth at 37°C, 180rpm is done. Following that the centrifugation of bacterial cells is done at 4000rpm for 10minutes at 4°C to get the supernatant. The resuspension is done for at least 1-2 minutes, and again in 0.1M MgCl2, 10ml of ice water. Another centrifugation is performed and the supernatant is again suspended in 3 ml of iced cold water with 0.1M CaCl2. Additional incubation is done for 2 hours, followed by another centrifugation at 4000rpm, 4°C, for 10 minutes. In the end, resuspension of the cells is done in 3ml of iced cold water, having 0.1 M of CaCl2 and 10% glycerol. This is stored in a small aliquot at around -80°C (Liu et al. 2018) Thus, the competent cells of E. coli are prepared that have more potential to take up genetic material like DNA.
Competent cells are at first incubated with plasmid or DNA on ice for 30 minutes. After that, these competent cells heat shock at 42°C for a minimum of 90 seconds in order to relax the cell wall. This accelerates DNA recombination as well as repair and when a chilled bacterial medium is added, there is a higher recovery rate of transformed cells. It is then incubated for 18-24 hours with ampicillin-like antibiotics to ensure resistance to antibiotics (Samuels, Drecktrah & Hall, 2018). Hence, if none of the cells dies that means there is the presence of transformed cells.
Agarose gel is taken using W/V percentage with the solution according to the appropriate concentration. Buffers such as TAE (containing 40mM of Tris-acetate and 1mM of EDTA), along with TBE that has 45mM Tris-borate as well as 1mM EDTA. This agarose along with the buffer is melted to completely dissolve the gel. After that Ethidium Bromide (EtBr) at a concentration of 0.5 microgram/ml is added. Sometimes dyes are added to stain the gel and after that electrophoresis are performed to separate the DNA produced after PCR, using Taq polymerase (Green & Sambrook).
“Pluthero’s method” is a useful way to extract Taq polymerase from the bacteria. This technique helps to reduce the production cost of this enzyme. The E. coli strains having the gene of Taq Pol 1 are harvested using centrifugation following 12 hours of incubation. After that, a buffer containing Tris-HCL, dextrose, with a pH of 7.9, EDTA as well as PMSF are used to wash the cells. Another centrifugation is performed followed by cell lysis using a buffer to help to get the DNA of the cells. Incubation and another centrifugation separate cell debris from the supernatant and in the end Taq polymerase is extracted (Chen et al. 2015).
Several experiments have stated that it is very important to perform DNAse treatment before PCR. Firstly, it significantly reduces the total number of DNA in the medium and eases up the attachment of Taq polymerase with DNA template. It is a very important step and if the enzyme cannot attach with the template it cannot precede with the extension step of PCR that results in the formation of two new DNAs (Green & Sambrook, 2018). DNAse ensures the destruction of excess DNA and accelerates the PCR process.
This test was done to determine the amount of Taq polymerase from E. coli and compare it with amount of commercial Taq polymerase. This research primarily separates the Taq polymerase and marks as purified Taq polymerase. Furthermore, this Taq polymerase was estimated with BCA protein analysis, which was nearly between the range of 0 to 0.5 mg/ml. Regarding this test, it was seen that using this assay with the commercial Taq was used to improve the density of the solution. However, regarding the outcome, it was seen that purified Taq polymerase provided a better result. In conclusion, it can be said that purified Taq polymerase is a better option than commercial Taq, which help in optimization of protein expression.
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