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In a molecular cloning experiment to determine the absence or presence of bacterial colonies on two plates, several observations were noted. The objective of this experiment was to transform a strain of bacteria, in this case, competent E. coli that had no antibiotic resistance through the insertion of DNA comprising of a gene which codes for resistance to the particular antibiotic. The results from the experiments demonstrated that indeed, E- coli had more than 1 level of gene regulation which allowed it to alter its phenotype quickly as it responded to environmental stimuli like for instance arabinose. This experiment was also conducted in order to determine whether there was successfully transfer of the gene into the organism. The presence or absence of the bacterial colonies was ascertained through the observation of the LB Amp plates. Form the results obtained; it was possible to ascertain the absence or presence of the bacterial colonies through close observation of the Lb/Amp plates.
In the results obtained from the experiment, it was proved that indeed, there was growth of E-coli in the plates and that the number of colonies were 51 and had confluent growth at the age. The growth was place yellow under normal light while under the Ultra Violet (UV) light, the color was Fluorescent Green. From the results obtained, it was also ascertained that there was successful transformation of the plasmids into the LB/amp/ara and LB/amp plates. The growth that was prevalent on the Ampicilin plates was a clear indicator that indeed the gene in the plasmid had been activated and thus there was successful transformation.
Various colours were depicted on the colonies of the LB/amp/ara plates under the UV light. For instance, the colour was Place yellow on the LB/amp plates while the color turned to fluorescent green on the LB/amp/ara plates when viewed under the ultra violet light. The ara C protein was proved to bind itself on the gene thus preventing gene activation hence leading to no gfp production. The ara C protein, was on the other hand known to bind itself on the Arabinose in the presence of the Arabinose thus allowing for activation of the gfp gene which eventually leads to the production of green fluorescence protein.
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Among some of the applications of genetic transformation in our daily lives includes the pharmaceutical products like for instance insulin, designing of test tube babies and in the production of pest/insect resistant crops like the genetically modified foods.
Due to its monotonous and long structure, it was difficult to analyze DNA as a molecule until in the 1970’s. Currently, it is now easier to analyze and excise a particular DNA region and produce several copies out of it as well as being capable of determining the DNA’s nucleotide sequence. Through the Biochemists located at the StanfordUniversity, it became clear that it was possible to transfer the genetic traits from one organism to another through molecular cloning. It was further ascertained that there was an edited exhibition of very specific modifications when the DNA of a given microorganism in the experiment was recombined with the Sequence of another DNA that was inserted.
One of the best approaches to achieve the above objective is through cutting the relevant gene from the DNA of a human being and splice or paste it in the plasmid DNA. The restriction enzymes, which happen to be over hundred, cuts in a precise manner the particular sequence of the molecule of DNA. Isolation of some part of the molecule for the Human DNA can be done because the distribution of such sites on the molecule of DNA is random. The introduction of Recombinant DNA technology has led to the discovery of various scientists of whole new classes of proteins and genes and concluded that indeed, many of the proteins are conserved highly in evolution. Further more, the Recombinant DNA technology has yielded new ways in which the individual domains in proteins and proteins in general functions. Thus, molecular cloning has made it easy to produce protein hormones and vaccines in bulk in order to produce substances which have economic and medical values (Zukowska, et al, 2006, p. 167).
Possible sources of error and limitations in the experiment
Some of the problems or limitations during the experiment were that the addition of more or less chemicals in the tubes containing the Plasmid DNA that was prepared could lead to wrong results. Picking of several instead of a single colony of bacteria from competent cell plates could also lead to unexpected results. Incubation of the tube for more or less time than expected in temperatures less or more that the recommended 42 degrees Celsius could also lead to results which are not accurate.
In general, some of the most common errors that can occur during the experiment include not using a RT control , application of inappropriate normalization control, poor probe and primer control, using of RNA that is of poor quality, lack of using master mixes, introduction of cross contamination, not setting the threshold and baseline properly, not performing melting or dissociation when using of the SYBR Green, poor efficiency of the reaction and use of inappropriate range for the standard curves.
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In the experiment, one can only be able to develop a single stranded DNA molecule having limited length despite the fact that it is possible to chemically synthesize DNA. This process is not only costly but also slow. Additionally not all the molecules of DNA can be replicated spontaneously once they are introduced into the cells. In order for molecular cloning to succeed, then several cloning vectors having key features should be included. Among them includes the origin of replication which must ensure that the genes are replicated together with the own cells of DNA, a selectable marker which will allow cells containing the vectors to be easily identified and selected for, multiple cloning sites to ensure that the gene of interest is easily inserted and the promoter sequence that is placed in the cloning vector to allow for gene expression (Hobbs, 2008, p. 211).
Vectors that are hybrid e.g. having two different species like the mammalian and bacterial are referred to as the shuttle vectors and they allow the movement of genes between the cells of two species which are different (Genecet Biotechnologies, 2008, p. 10). .
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Suggested improvements to remove the errors or limitations in the experiment
In order to minimize or completely remove any errors or limitations that can arise in the experiment, then it is vital to consider making some improvements. Among such improvements include the use of exact or relevant measurement of the required chemicals in order to ensure accurate results. The proper use of both materials and chemicals will ensure that accurate results are obtained from the experiment. Furthermore, the incubation of the solutions contained in the LB should take the appropriate recommended time since taking less time or more time during the process of incubation of such chemicals will result in inaccurate results from the experiment.
The double helical structure’s identification of DNA has opened the entire world to a new era of both biological research and understanding. Scientists, trough understanding the molecular structure of the Molecule of DNA are now capable of manipulating its function. This is done through joining two pieces of DNA through a technique known as ligation. Ligation eventually results into a plasmid that is finally inserted into another living organism through a procedure referred to as genetic transformation. During the production of any recombinant DNA, both techniques are widely used by scientists. Ampicilin is used in the selection of positive transformants because it is a resistant gene that is included in the vector of cloning. In order to transform the whole organism genetically, then one must insert the new genes in each single cell of the organism through a process known as Total Genetic Transformation (Watson, 2011, p. 50).
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