Gene Technology: Practical Report
Gene technology, also known as genetic engineering, involves manipulating or transferring genetic material within or between organisms. It has the potential to increase agricultural yields and reduce the use of pesticides. This report aims at examining two experiments conducted on cloning of genes through electroporation and DNA cloning as well as induction of GFP protein.
The report is divided into three segments. The introduction defines some of the key terms used end to end in the report. In the second part, the two experiments are outlined and the information provided analyzed thereby discussing the results in the same section. In the last section, the main points are highlighted.
Keywords: electroporation, DNA Cloning, GFP Protein
Before the writer proceeds to discuss the results of the two experiments, it is paramount to define the key terms used throughout the essay. One of the terms is electroporation, which refers to the method of changing DNA and more specifically, for plants whereby high voltage pulses of electricity open up pores in cell membranes such that the foreign DNA can pass. Electroporation technique could be used in a number of applications including the establishment of plasmids into the living cells for transfection, fusion of cells and also induction of proteins into the cell membranes. In medicine, electroporation has been of considerable importance in catalyzing the intakes of DNA by the protoplasm. Further, electroporation is used in treating of cancerous cells. In this case, electric pulses are placed in a tumor so as to catalyze the ability of anti cancer drugs to enter tumor cells. In short, this process enhances permeability of the cell membranes so that foreign DNA may pass through (Phillips, 2012).
DNA Cloning, on the other hand, refers to the creation of similar copies of a single gene. This is usually done with the purpose of having sufficient material for further research. The copies of the DNA molecules are known as clone libraries. With the advance in genetic technology, it is possible to obtain copies of the entire cell in an organism just like the normal cell division process. In this case, the copies are known as cell lines, which should in ideal conditions, are similar to the original cell. Cloning has also been used to produce identical organisms including animals like the popular Scottish sheep (Lefers, 2004).
GFP is the short form for Green Fluorescent Protein that is obtained from the Jellyfish. It emits a bright green fluorescence and is used to monitor gene expression and transfer across the cell membrane (Mosby, 2009).
Green Fluorescent protein is of considerable importance in addressing several issues in single cells, as well as tissues in the organisms. Besides, individual clones can be generated from single cells
Overview of the First and the Second Practicals
Bacteria are microorganisms which comprises of minute DNA molecules called plasmid that naturally duplicate into identical molecules by natural means. Biotechnologists have artificially cloned the DNA molecules so as to increase the material for further research. In fact, the plasmid can be duplicated into similar plasmids independently of the host. The plasmids also are remarkably resistant to antibiotics, but biotechnologists use electroporation technique so as to enhance permeability of the antibiotics. As highlighted above, green fluorescent protein originates from the jelly fish and is visualized using ultra violet light after cloning it with the bacteria
In addition, each plasmid has a multiple cloning site where restriction enzymes can achieve their function. Biotechnologists clone the gene of interest using ligation enzymes, and introduce this recombinant product into bacteria using electroporation, so that the gene of interest can be expressed. One of the techniques to examine the protein of interest is the Western blot that comprises of GFP cloning as well as the induction and expression of the GFP protein.
Introduction to Practical A
The first practical was carried out over two weeks such that, electroporation was done in the first week while, DNA cloning commonly known as replication was done in the second week. The practical aimed at transforming the GFP genes into E.coli.
Aims of the Experiment
The main aim of the first experiment was, to transfer PGFP and PBCKS into competent bacteria using electroporation technique and scan for antibiotic by plating these bacteria on two groups of lab plates.
Results and Discussions
From the results obtained by group three, It is clear that there was significant growth of the bacteria as expected. The two plates had been exposed to ultra violet light where two colonies were observed including that of the white colonies as well as the green colonies. However, this is not always the case. At times, the electroporator may be defective and, therefore, there may be unobservable results from the experiment. In this case, the white colonies observed were labeled as PBCKS while the green colonies indicated as PGFP.
The results provided from the group indicated a significant growth on the plates. This was an indication that the plasmid DNA was transformed into bacteria as was expected. Before the experiment was conducted, the electroporator was checked to ensure it was working. The plates on the other hand, the plates were incubated at the right temperatures favorable for the growth of bacteria. In addition to this, the LB liquid was added as soon as possible after electroporation. From observations, both white and green colonies were seen and, therefore, it was appropriate to note that the GFP gene was expressed as green hence the colonies were observed as green in color.
Introduction to Practical B
The second part, as mentioned earlier, involved DNA cloning. Its main aim was to isolate the two colonies from bacteria and digest them using the restriction enzymes to get sticky ends and linear DNA fragments. There are a number of possibilities. One of the possibilities is that the GFP gene rejoins and returns to its place on its plasmid and this indicates no DNA recombination. Under such circumstance, no growth may occur on the plates. Secondly, the DNA fragments of the PBCKS may rejoin their plasmid and no DNA recombination happens. In case the bacteria take up this plasmid, the bacteria will ultimately grow on the plate since the bacteria became resistant to chloramphenicol. In this case, if the plates are exposed to Ultra violet light, the PBCKS will appear on the plate. Lastly, the GFP gene may be litigated to PBCKS thereby the bacteria taking up the plasmid. Similarly, growth would appear on the plate that has chloramphenicol since the bacteria would be resistant to the antibiotic. If the plate is exposed to ultra violet light, some green colonies would be observed, therefore, showing the expression of the GFP.
Results and Discussions
In this experiment, the plates were clearly observed where one of the plates indicated the presence of white colonies when subjected to ultra violet light. The white colonies were observed thereby indicating that bacteria, indeed grew since they became resistant to chloramphenicol.
In the second plate, green color was observed indicating the presence of the green colonies labeled as PGFP. This was observed after the plate was subjected to ultra violet light and therefore, showing the expression of the GFP.
Introduction to the Second Practical
The second practical involved the induction and expression of GFP protein in E.coli whose goal was to induce the expression of GFP in E.coli by use of IPTG at different times. The core aim was to determine protein concentration at any given time. A BioRad protein dye was used which usually depends on change in color. The practical was also aimed at separating protein mixtures from cells. In line with this, the GFP were to be separated from a mixture of proteins with respect to molecular sizes.
Aim of the Practical
The practical aimed at testing the presence of GFP by use of the western blotting method and also by use of antigens antibodies reaction.
Results and Discussions
From the practical, it was found out that there subsisted a direct relationship between protein concentration and incubation time. In fact, protein concentration increases radically with time. In addition, it was possible to separate GFP from other proteins according to the molecular weight. Besides, the GFP was tested and detected by use of antigens antibodies reactions and for sure, it was found to be the GFP.
In the second practical, as indicated, was aimed at determining the protein concentration at different times. The protein concentration could be plotted against time so as to determine the concentration of proteins at various time intervals. The following results were obtained. It was discovered that the protein concentration increased gradually with time.
TIME 1 2 3 4 5 6 7 8 9 10 11 12
EXPER 1 0.328 0.332 0.335 0.342 0.346 0.345 0.34 0.345 0.34 0.345 0.337 0.345
EXPER2 0.344 0.348 0.476 0.478 0.459 0.61 1.058 0.91 1.242 1.149 1.638 1.627
EXPER 3 0.417 0.532 0.453 0.417 0.414 0.397 0.759 0.844
Calculations are attached at the end of the report in an excel document.
From the above graph, assuming the mean values are on the Y axis and the time on the X axis, then upward sloping line from left to right shows a direct relationship between protein concentration and time. In this case, the protein concentration increases gradually with time.
Further, if the mean values are plotted against time, the following graph would be obtained.
If a line of best fit is obtained, similar results of positive correlation would be established.
From the above practical, it is clear that indeed, genes can be multiplied or rather replicated into identical genes. To validate this, the practical indicated growth of bacteria within a period of two weeks. This was made possible through gene cloning. In fact, gene cloning has been useful when it comes to the preparation of material for further research. Indeed, research is quite expensive, and the biotechnologists have come up with the idea of cloning so as to produce more material to carry out research.
Electroporation, on the other hand, has been used to open up pores in cell membranes so that foreign DNA can pass through it. This technique has been used in quite a number of applications which includes the introduction of plasmids into living cells, as well as induction of proteins into the cell membranes. In short, this process enhances permeability of the cell membranes so that foreign DNA may pass through it.
The growth of bacteria can be clearly observed when the plates are subjected to ultra violet light. In case there is no growth, then nothing would be observed on the plates, and this could be caused by a number of factors including faulty electroporator.
The second practical clearly indicated a correlation between the protein concentrations and time. It was clear that protein concentration increased gradually with time. Besides, it was crystal clear that proteins can be separated from each other according to their molecular size. With regards to GFP, it is relatively easy to recognize it by use of antigens antibodies reactions.
In order to achieve sensible results, it is essential to ensure that the correct procedures are followed; the equipments are properly used and checked before use.
Lefers, M. (2004). Cloning. Holmgren Lab Journal, 1-4.
Mosby. (2009). Medical Dictionary. Elsevier.
Phillips, T. (2012). Electroporation. Biomedical Journal, 1-6.